Decrypting the programming of β-methylation in virginiamycin M biosynthesis

Decrypting the programming of β-methylation in virginiamycin M biosynthesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Decrypting the programming of β-methylation in virginiamycin M biosynthesis Sabrina Collin, Russell Cox, Cédric Paris, Christophe Jacob, Benjamin Chagot, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2103032/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract During biosynthesis by multi-modular trans-AT polyketide synthases (PKSs), polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase (HMGS) cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT PKS. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation, identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, imperfections in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nature deploys an assembly line strategy to construct polyketide specialised metabolites, in which each task is assigned to a specific enzyme. In the prototypical cis -AT systems, the majority of these functions are present within catalytic domains of gigantic multienzymes called polyketide synthases (PKSs) 1 . The functional domains are clustered into modules, where each module is typically responsible for carrying out one round of chain extension and b-processing of the resulting intermediate. In addition to the three domains which are essential to chain building (acyl transferase (AT), ketosynthase (KS), and acyl carrier protein (ACP)), many modules also harbour optional domains which modify the oxidation state of the b-keto group resulting from the condensation reaction. The division-of-labour organisation of PKS systems makes them attractive targets for synthetic biology approaches aiming at generating high-value derivatives 2 . Relative to the cis -AT PKSs, the trans -AT systems 3 ( Fig. 1a ) incorporate one or more free-standing enzyme activities and a wider variety of enzymatic functions, including cassettes of enzymes which introduce b-branching into the polyketide intermediates 4 . A common modification is b-methylation, which involves five discrete proteins ( Fig. 1b ): (i) a malonate-loaded ACP (called ACP donor, ACP D ); (ii) a condensation-inactive KS domain (KS 0 ) which generates acetyl-ACP D from the malonyl-ACP; (iii) a 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) homologue which catalyses attack of the acetate-derived nucleophile on the b-keto group of the polyketide chain attached to an acceptor ACP (ACP A ), yielding an HMG- S -ACP A thioester; (iv) an enoyl-CoA hydratase (ECH) homolog (ECH 1 ) that serves as a dehydratase to produce the corresponding a,b-unsaturated thioester; and finally, (v) a second ECH homolog (ECH 2 ) that catalyses decarboxylation to afford the b-methyl product. Variation of the electrophile and nucleophile structures, and/or HMG processing sequences, gives access to further types of b-functionality 4,5 . An intriguing feature of b-modification is how the system selects which polyketide-ACP A intermediate to target, as every round of chain extension yields a potential b-keto substrate. In principle, gate-keeping by the HMGS would be sufficient to direct the whole cassette, as no downstream enzymes can act in the absence of this chemistry 6 . Previous work identified a sequence motif including a conserved Trp flag characteristic of ACP domains in modules targeted for b-methylation 7 . As the majority of the residues are confined to the domain core, a model was proposed 7,8 in which burial of the Trp side chain gouverns the orientation of helices a2 and a3 within the ACP four a-helix bundle, allowing both specific residues on helix a3 and the substrate to interact with the HMGS. Nonetheless, the detailed recognition mechanism remains obscure, hampering efforts to install b-branches at will by redirecting HMGS cassettes to alternative ACP A -bound intermediates. Here we aimed to understand the control of b-methylation during biosynthesis of virginiamycin (Vir) M 1 by a hybrid trans -AT PKS-nonribosomal peptide synthetase (NRPS) system 9 ( Fig. 1a ). In this case, b-modification was thought to occur exclusively during chain extension by module 5 (M5) ( Fig. 1ab ) 3 . As is typical of b-branching modules 8 , Vir M5 incorporates a tandem of ACP A domains, ACP 5a and ACP 5b . We show by Trp fluorescence quenching that the Vir HMGS, ECH 1 and ECH 2 homologues (VirC−VirE) preferentially bind ACP 5b in its holo and substrate mimic forms, consistent with in-series function in which ACP 5a participates in chain extension and ACP 5b in b-methylation 10 . Comparative structural analysis of multiple Vir and mupirocin ACPs at high-resolution reveals essentially identical folds, excluding helix a3 orientation as the basis for specific recognition. Instead, the crystal structure of the holo -ACP 5b −VirD complex identifies an ACP interaction motif centred on the phophopantetheine (Ppant) prosthetic arm and surrounding secondary structural elements, with specificity conferred via distinctive electrostatic surface features of the domain combined with precise Ppant positioning. We also show in vitro that Vir holo -ACP 7 is recognised by the cassette enzymes, and identify a doubly b-methylated Vir M derivative in production extracts. The ca. 150-fold lower titres of the analogue relative to Vir M 1 imply that this second b-methylation is suppressed in vivo . Taken together, our data show that b-methylation programming relies on at least two distinct control mechanisms, but remains imperfect, identifying the deactivation of such measures as a promising strategy for polyketide analoguing. Results And Discussion Binding of VirC−E to Vir ACPs. We first assessed the interaction between recombinant apo - and holo -ACP 5a and ACP 5b and VirC−VirE from the Vir pathway of Streptomyces virginiae 9 , with ACPs 6 and 7 from non b-methylation modules selected as controls in vitro ( Extended Data Figs. 1 and 2 , Supplementary Tables 1 and 2 ). While the apo forms are not physiologically relevant, the holo proteins are present at several stages of the catalytic cycles 11 . No binding was detected using tryptophan fluorescence quenching 12 between VirC−VirE and holo -ACP 5a , although apo -ACP 5a was weakly bound (K d = 166 ± 18 mM, 102 ± 14 mM and 653 ± 171 mM, respectively) ( Extended Data Table 1, Supplementary Figs. 1 − 3 ). While interaction with apo -ACP 5b was similarly weak (77 ± 10 mM, 178 ± 26 mM and 171 ± 31 mM), the three enzymes showed good affinity towards holo -ACP 5b (3.8 ± 0.5 mM, 2.9 ± 0.5 mM and 6.8 ± 1.5 mM), consistent with an important role in recognition for the Ppant arm. To provide a more native context to these assays, we also analysed binding of VirC−VirE to the holo -ACP­ 5a -ACP 5b didomain ( Extended Data Figs. 1 and 2 ). The observed binding affinities (K d = 17 ± 2 mM, 19 ± 3 mM and 42 ± 10 mM) ( Extended Data Table 1, Supplementary Figs. 1 − 3 ), are within the same order of magnitude as for the discrete holo domains, and thus we find no evidence for cooperative binding of tandem ACP A s as previously proposed 13 . Furthermore, no binding was detected by VirC−VirE to control ACP 6 in either its apo or holo forms. However, unexpectedly, both apo - and holo -ACP 7 behaved similarly to the analogous forms of ACP 5b with VirC−VirE ( apo -ACP 7 : 75 ± 7 mM, 75 ± 7 mM and 301 ± 126 mM; holo -ACP 7 : 18 ± 3 mM, 4.3 ± 0.2 mM and 22 ± 5 mM) ( Extended Data Table 1, Supplementary Figs. 1 − 3 ). Finally, we evaluated binding of VirC−VirE to ACP 5a and ACP 5b modified, albeit imperfectly, to mimic the native substrates 14 ( Extended Data Fig. 2 ): acetoacetate (VirC), ( RS )-3-hydroxy-3-methylglutarate (VirD) and 3-methylcrotonate (VirE). The trends in relative affinities for acetoacetyl-ACP 5a / 5b and methylcrotonyl-ACP 5a / 5b were in line with those for the holo proteins ( Extended Data Table 1, Supplementary Figs. 1 − 3 ), consistent with the strong preference of the cassette for ACP 5b , while the presence of substrate analogues did not increase but moderately diminished affinity. The latter result, which implies substrate tolerance, is encouraging for the prospect of generating analogues. Binding by VirD to HMG-ACP 5a was likewise weaker than to HMG-ACP 5b , although the difference was less marked than for the other analogues (51 ± 25 mM vs. 39 ± 5 mM) ( Extended Data Table 1, Supplementary Fig. 2 ). We excluded an effect on binding to HMG-ACP 5b of catalysis, by testing a catalytically-inactive version of VirD (E128Q 15 ) ( Extended Data Fig. 1 , Supplementary Fig. 2 ), which yielded essentially the same K d (33 ± 3 mM) as for the wild type protein. Overall, the bulk of the fluorescence quenching data are consistent with preferential binding by the three b-methylation enzymes of ACP 5b . Characterisation of the ACP 5b /cassette interactions. To gain insight into the determinants of interaction specificity, we aimed to solve the structures of complexes of ACP 5b with VirC−VirE. However, we were unable to obtain crystals with wild type recombinant VirC and VirE ( Extended Data Fig. 1 , Supplementary Tables 1 and 2 ), nor with VirC quadruply mutated to promote crystallisation (C114A/Q334A/R335A/R338A) ( Extended Data Fig. 1 ), as previously described for its homologue CurD from the curacin pathway 13 . Nonetheless, comparison of small-angle X-ray scattering (SAXS) data obtained on wild type VirC complexed with holo -ACP 5b , with that calculated 16 from the crystal structure of the acetyl-ACP D −CurD complex (PDB: 5KP6) 13 , revealed a remarkable fit between the experimental and theoretical scattering curves (c 2 = 1.524) ( Extended Data Fig. 3 , Supplementary Table 3 ). This result shows that the overall structures are similar, implying that the ACP/partner recognition elements are likely to be shared between the two complexes. In the acetyl-ACP D −CurD case 13 , the interface involves the entirety of helix a2, the loop a2-a3 and helix a3, as well as a key orientational interaction between the Ppant phosphate and CurD Arg33 13 . Next, we successfully solved the structure of VirD alone at 1.7 Å resolution (PDB ID: 8AHZ) ( Fig. 2a , Supplementary Table 4 ), as well as that of the holo -ACP 5b −VirD complex by Se-SAD at 2.1 Å (PDB ID: 8AHQ) ( Fig. 2b ). The final VirD model consists of a trimer in the asymmetric unit with r.m.s.d. between monomers of 0.2 Å (202 C a ), whose solution relevance was confirmed by SAXS analysis (PRIMUS 17 ) ( Extended Data Fig. 3 , Supplementary Table 3 ). VirD belongs to the crotonase superfamily whose members exhibit a characteristic fold formed from repeated ββα units 18 ( Fig. 2 ). In the holo -ACP 5b −VirD complex (PDB ID: 8AHQ) ( Fig. 2b ), the asymmetric unit contains two monomers of VirD and two of holo -ACP 5b . As determined by the H3 crystal symmetry, VirD forms characteristic homotrimeric disks 18 , two of which are stacked, with six ACPs distributed equatorially at the interface between the trimers. In this arrangement, the smallest gap between S6871 of ACP 5b bearing the Ppant (~20 Å) and the catalytic E128 of a VirD monomer is ca. 13.9 Å ( Fig. 2c ), with the other VirD active sites more than 33.8 Å distant. VirD elements contributing to the interface include the b-strand b10 and the subsequent loop (b10-a4) of one monomer, and the b-turn (b1−b2), the loop (a1−b4) and helix a7 of a second monomer ( Fig. 2bc ) . Notably, the interface also incorporates the well-folded helix a10 of the first monomer, which is disordered in the structure of VirD alone ( Fig. 2a ). Concerning the ACP, the interaction involves the C-terminal portion of helix a1, the adjacent loop (a1−a2) and the N-terminal regions of helices a2 and a3. Specific interface residues include T6850 (helix a1), Y6852 (loop a1−a2), D6870, I6872, V6875 and E6876 (helix a2) and Y6895 (helix a3) ( Fig. 2c , Extended Data Fig. 4 ). The structure of the complex also identifies key interactions between VirD and the Ppant tethered to S6871 of ACP 5b (the distal end of which is not visible in the electron density ( Fig. 2c )), consistent with its contribution to binding affinity as observed by fluorescence quenching ( Extended Data Table 1 ). Specifically, R125 of VirD, whose side chain is oriented by a water molecule, forms a salt bridge with the Ppant phosphate. The same water molecule bridges R192 from an adjacent VirD monomer, which participates in a salt bridge with D6870 of ACP 5b . The opposite end of the D6870 carboxylate sits in an oxyanion hole comprising the NH groups of I6872 and L6873 of ACP 5b helix a2. Overall, these interactions place the oxygen of S6871 within 13.9 Å of the buried catalytic E128 of VirD, and thus within reach of the Ppant arm. Finally, we turned our attention to VirE, studying its interaction with holo -ACP 5b in solution by SAXS combined with modelling using AlphaFold 19 ( Extended Data Fig. 3 , Supplementary Table 3 ). Globally, this analysis indicates that VirD and VirE exhibit essentially the same overall folds and trimeric quaternary structures, and thus accordingly, the observed holo -ACP 5b −VirE complex closely resembles that of holo -ACP 5b −VirD. Structural basis for ACP 5b /VirD interaction specificity and ACP anti-selection . Identification of the amino acids in ACP 5a corresponding to the ACP 5b interface residues shows that with only one exception (V6749 [ACP 5a ] vs. T6849 [ACP 5b ]), they are identical ( Extended Data Fig. 4 ). Thus, this set of residues does not constitute the basis for specific recognition of ACP 5b by VirD. We therefore reassessed the hypothesis 7 that ACP recognition might derive, at least in part, from the relative orientation of the a-helices within the domain structures. For this, we solved the NMR structures of holo -ACP 5a (PDB ID: 8A7Z), holo -ACP 6 (PDB ID: 8AIG) and holo -ACP 7 (PDB ID: 8ALL) ( Supplementary Table 5 ), complementing the previously solved apo -ACP 5b (PDB ID: 4CA3) and apo -ACP 5a (PDB ID: 2MF4) structures 10 . Superimposition of holo -ACP 5b (PDB ID: 8AHQ) from the holo -ACP 5b −VirD crystal structure ( Fig. 2b ) with apo -ACP 5b , apo -ACP 5a , holo -ACP 5a , holo -ACP 6 and holo -ACP 7 reveals r.m.s.d. of 0.732 Å (74 Ca), 1.076 Å (59 Ca), 1.095 Å (72 Ca), 1.781 Å (72 Ca), 2.734 Å (72 Ca), respectively. ACPs 5a, 5b, 6 and 7 thus exhibit the same overall organisation including the orientation of the four a-helices ( Fig. 3a ), an architecture conserved with the previously-characterised Mup ACPs on which the Trp flag model was based 7 ( Fig. 3b ). Therefore, while the Trp provides strong predictive value for ACP sites of b-branching 7 , our results argue against an important role for this residue and the resulting orientation of helix a3, as determinants of cassette interaction specificity with ACP A s. Indeed, both ACP 5a and ACP 5b contain Phe at this position instead of Trp. This observation is in line with further sequence variability recently uncovered at this position in other trans -AT PKS systems ( Extended Data Fig. 4 ) 20–23 . The origin of the observed minor differences in r.m.s.d. lies in the positions of the main chains of the loop regions, particularly a1−a2. This observation prompted us to consider the potential contribution of the a1−a2 loop to recognition. Gratifyingly, close inspection of the holo -ACP 5b −VirD complex structure ( Fig. 2c ) identified ACP 5b N6865 located in the a1−a2 loop as a potential specificity determinant. The d-oxygen and nitrogen atoms of N6865 hydrogen bond to two water molecules which are members of a larger, four-molecule water network forming hydrogen bonds to the main chain atoms of ACP 5b residues N6865, L6869, D6870, L6873, and L6894. The constraints imposed by this network on the L6869 carbonyl, coupled with those on the D6870 side chain resulting from interaction with R192 of VirD and the ACP helix a2 oxyanion hole, position the D6870 carboxylate at a distance of 3.9 Å from the phosphate of the Ppant arm. The resulting position adopted by the Ppant to minimise electrostatic repulsion with D6870 apparently favours its efficient interaction with VirD. Notably, in ACP 5a , polar N6865 is substituted by hydrophobic L6764 ( Extended Data Fig. 4 ), a residue which cannot participate in the hydrogen bond network. Nonetheless, analysis of the ACP 6 and ACP 7 sequences reveals that the situation is more complicated than is evident from a single complex structure, as the equivalent sequence position in ACP 6 that does not interact with VirD is a Glu, while that in ACP 7 which is recognised, is also a Leu ( Extended Data Fig. 4 ). Thus, if a comparable water-mediated hydrogen bonding network is necessary to establish the correct orientation of the Ppant for binding VirD, other residues in ACP 7 can apparently substitute for the Asn of ACP 5b . Inspection of the ACP structures also revealed that they diverge in terms of the pattern of charged, hydrophilic and hydrophobic residues on the surfaces adjacent to the Ppant arm ( Fig. 4 ) 24 . Notably, in the case of ACP 5b ( Fig. 4a ), the surface surrounding the negatively-charged phosphate group of the Ppant and the adjacent, conserved acidic residue D6870, is largely hydrophobic but punctuated by a protruding hydrophilic region composed of S6863, N6865 and T6866. This region is itself encircled by three acidic patches, two contributed by the a1−a2 loop (E6854, D6857; D6859, E6861), and the third located at the N-terminus of helix a3 (D6896). ACP 7 exhibits an overall similar charge distribution to ACP 5b ( Fig. 4b ). In this case, the hydrophilic patch is replaced by closely co-localised residues R2004, L2007 and E2008, while the ACP 5b acidic patch comprising D6859 and E6861 is maintained by ACP 7 ­ residues D2001 and D2003. The surface additionally comprises an acidic residue D1995 unique to this domain. In contrast, in ACP 5a ( Fig. 4c ), the hydrophilic protrusion is less extensive, and flanked by a hydrophobic region comprising F6763 and L6764, while one of the equivalent a1−a2 loop patches contains the positively-charged R6756. In addition, residue A6862 in ACP 5b is replaced by E6761 in ACP 5a , contributing an additional negative charge to the surface ( Extended Data Fig. 4 ). Consequently, when the residue at this position is small and hydrophobic it can participate in the ACP core, but when charged, the side chain points towards the solvent. ACP 6 differs even more dramatically from ACP 5b ( Fig. 4d ). Specifically, the hydrophilic cluster is replaced by the acidic residue E1218 which is sandwiched between two cationic amino acids, R1206 and R1250, and uniquely among the four ACPs, the domain contains an additional positively-charged residue R1228 near the Ppant phosphate. Thus, both ACPs 5a and 6 exhibit positive net charge in regions which are negatively-charged in ACPs 5b and 7, electrostatic features which we propose disfavour productive complex formation with the b-cassette enzymes. Site-directed mutagenesis supports the specificity model. Taken together, the obtained data suggested a model in which ACP recognition by VirD (and possibly all cassette members) depends principally on the subtle electrostatic landscape of the ACP surface which drives certain interactions, and potentially on the precise positioning of the Ppant arm within the resulting binary complexes, with only a minor role played by the attached substrates. To directly test this idea, we exchanged a1−a2 loop residues E6761 and L6764 of ACP 5a with their equivalents in ACP 5b , A6862 (position contributing to the surface potential) and N6865 (surface hydrophilicity and/or Ppant orientation) ( Extended Data Figs. 1 and 2 , Supplementary Table 1 ), and evaluated binding of the single and double mutants to VirD by tryptophan fluorescence quenching. While VirD failed to bind holo -ACP 5a , it showed good affinity to both of the single holo -ACP 5a mutants (E6761A [6.1 ± 0.6 mM] and L6764N [4.1 ± 0.4 mM]), with K d s comparable to those for binding holo -ACP 5b ( Extended Data Table 1 ). Thus, either single mutation results in VirD recognition. Binding to the double mutant was also observed (7 ± 1 mM), albeit at slightly reduced affinity, perhaps due to minor perturbation of the ACP 5a structure as judged by circular dichroism ( Extended Data Fig. 1 ). It is also notable that VirD systematically exhibited higher affinity for the holo form of the ACP 5a mutants relative to the apo forms (by 4−20-fold) ( Extended Data Table 1 ), confirming the crucial role of the Ppant cofactor in the interaction. We also demonstrated that, in contrast to the lack of binding of wild type holo -ACP 5a by VirC, both the E6761A and L6764N holo -ACP 5a single mutants were recognised (4.7 ± 0.5 mM and 11 ± 1 mM, respectively), while affinity to the double mutant was on par with that of L6764N (10 ± 1 mM) ( Extended Data Table 1 ). These data support the idea that the a1−a2 loop region of ACP 5b­ is also critical for its preferential recognition by VirC. Although the ACP 5b −VirC complex evidently resembles that of ACP D −CurD, understanding the detailed role played by these residues in the interaction awaits higher resolution structural information. Identification of a doubly b -methylated virginiamycin derivative. The observed binding between ACP 7 and the VirC−VirE implied that the ACP 7 -tethered intermediate may be targeted by the b-methylation cassette in vivo . To evaluate this idea, we scrutinised extracts of a second virginiamycin-producing strain, Streptomyces pristinaespiralis ATCC 25486 (Sna cluster, Fig. 1 ). In contrast to S. virginiae , the complete genome sequence of S. pristinaespiraelis is available, which is a necessary prerequisite for using CRISPR-Cas9 25 to verify the genetic origin of any detected metabolites, while avoiding off-target effects. To demonstrate the relevance of our interaction studies to this second strain, we measured binding between recombinant ( Extended Data Figs. 1 and 2 , Supplementary Tables 1 and 2 ) apo - and holo -Sna ACP 7 and VirD. The sequence of VirD shows 63% identity to its Sna homologue, SnaJ 26 . Reassuringly, the K d determined for the most relevant holo form (13 ± 1 mM) was essentially identical to that measured for holo -Vir ACP 7 , while that for the apo -form was 2-fold lower (166 ± 25 (Sna) vs. 75 ± 7 (Vir) mM) ( Extended Data Table 1 ). Next, LC-HRMS analysis of S. pristinaespiraelis extracts revealed a signal at m / z = 526.2912 ([M+H + ]) (rt = 13.56; Fig. 5 ), in excellent agreement with the calculated for potential analogues of Vir M 1 , incorporating a second b-methyl at C-16 ( 3 , Fig. 1c ). Masses corresponding to alternative doubly b-methylated metabolites were not detected ( Extended Data Fig. 5 ). Importantly, 3 was no longer detectable in S. pristinaespiralis extracts when a portion of the module 7/module 8 interface was deleted using CRISPR-Cas9 ( Extended Data Fig. 6 , Supplementary Fig. 5 ), directly confirming 3 as a product of the Sna pathway. Using commercial Vir M as a reasonable calibration standard ( Supplementary Fig. 4 ), we estimated the titres of 3 at 150−200-fold reduced relative to 1 and 2 ( Extended Data Table 2 ). Therefore, while Vir ACP 7 is recognised with good affinity by the b-methylation cassette, this modification is reduced under native biosynthetic conditions ( Fig. 1a ). As the low absolute yields of 3 ( Extended Data Table 2 ) precluded purification, to further support the structural assignment, we carried out comparative MS 2 analysis of 1 − 3 ( Extended Data Fig. 7 , Extended Data Table 2 ), and fed S. pristinaespiralis cultures with isotopically-labelled amino acids, both individually and in combination: L-proline-2,5,5-D 3 , L-serine-2,3,3-D 3 and L-proline-2,5,5-D 3 + L-serine-2,3,3-D 3 . These amino acids were selected to confirm the relatedness of 3 to 1 and 2 ( Fig. 1a ), and simultaneously track the post-incorporation chemistry via loss of deuterium. Comparison of the feeding data obtained on 3 to those of 1 and 2 ( Extended Data Fig. 8 , Supplementary Fig. 6 ) demonstrates that 3 incorporates both Ser and Pro residues. Furthermore, the pattern of incorporation into 3 is consistent with retention of two deuteriums from both Ser and Pro. More specifically, the observed Pro labelling provides evidence for incorporation of L-proline-2,5,5-D3 followed by dehydrogenation, as in 1 ( Fig. 1c ). To explain the divergent labelling from Ser, we propose that Ser is incorporated by module 8 as normal, but that the subsequent HC-catalysed heterocyclisation/dehydrogenation does not occur due to mismatched substrate specificity. Indeed, the obtained MS 2 data are consistent with a structural difference between 1 and 3 in this region ( Extended Data Fig. 7 , Extended Data Table 2 ). Proline is then added by module 10, the product is liberated from the assembly line by macrocyclisation, and the Pro undergoes the native dehydrogenation reaction. Finally, transformation of the Ser to dehydroalanine may be catalysed spontaneously by an adventitious cellular enzyme, explaining the loss of the C-2 proton but retention of the two labels at C-3. Indeed, minute quantities of compound potentially corresponding to the non-dehydrated metabolite ( m / z = 544.3017; rt = 10.49 min) are also observed ( Extended Data Fig. 5 ). Conclusions Diverse b-modification reactions occur during biosynthesis by many trans -AT PKSs, and certain cis -AT systems 3,27,28 . How specificity is achieved is an intriguing question, as the b-modification cassette enzymes must distinguish between a large number of ACP A domains bearing potential b-keto substrates. A further layer of complexity is the typical presence in b-branching modules of 2−3 acceptor ACPs (ACP A s), implying that one or all of these domains could serve as the site for the reaction series 29 . Understanding how acyl-ACP substrates are chosen, or conversely counter-selected, is a prerequisite to introducing b-modification reactions at specific alternative positions in polyketides by genetic engineering. In this work, we investigated the b-methylation module 5 present in the virginiamycin (Vir) trans -AT PKS-NRPS, which comprises a KS domain and tandem ACPs (ACP 5a and ACP 5b ) ( Fig. 1a ). Both ACP domains contain a Trp to Phe substitution at a residue position previously proposed to be critical for flagging the trans -AT PKS ACP A s at which b-modification should occur 7 ( Extended Data Fig. 4 ), raising the questions of how they are recognised by the Vir cassette enzymes. Furthermore, the higher accessibility of ACP 5b as revealed by the module 5 SAXS structure 10 , suggested that it might be the preferred site of b-modification in trans . We show here that all members of the b-methylation cassette do indeed preferentially recognise ACP 5b , even when excised from its modular context, and that b-modification occurs within defined ACP 5b /partner complexes ( Fig. 2 , Extended Data Fig. 3 ). The gate-keeping function within the cassette is therefore not limited to the HMGS VirC. The crystal structure of the holo -ACP 5b −VirD complex ( Fig. 2bc ) in combination with comparative sequence analysis ( Extended Data Fig. 4 ), further reveals that the key ACP 5b interface residues are highly conserved with ACP 5a . ACP 5b selectivity instead derives in large measure from the electrostatic character of the surrounding amino acids which drive complex formation 30 ( Fig. 4 ). Hydrogen-bonding restraints imposed on the Ppant cofactor ( Fig. 2c ) may additionally optimise ACP/cassette interactions, but the attached substrates appear to contribute only minimally to the binding affinity 13 ( Extended Data Table 1 ). Overall, this specificity for ACP 5b likely ensures that two ACPs act principally in-series to support, respectively, chain extension and b-modification, which contrasts with previous reports of in-parallel function for such tandem ACPs 27,29,31 . This mechanism would require that the b-keto intermediate be transacylated between the two ACPs, a transfer that is compatible with the measured inter-ACP distance 10 . It would also necessitate that the holo form of ACP 5b be present, but there is precedent for this in trans -AT PKS systems 32 . While we can only speculate as to possible explanations, holo -ACP 5b may not be an efficient substrate for malonylation by the trans -acting AT (VirI/SnaM), and/or it may be poorly accessible to the AT due to preassembly of complexes between ACP 5b and the b-methylation cassette enzymes. Given the prevailing view in the literature that b-modification occurs with high fidelity 8 , we were surprised to observe that ACP 7 is also efficiently recognised by the three cassette enzymes in vitro , an interaction which translates in vivo into a previously-unidentified Vir M analogue 3 bearing a second b-methyl group. Notably, titres of 3 at ca. 1% of those of 1 , are on par with amounts of polyketides typically obtained by PKS genetic engineering 2 . While this result might be interpreted as indicating that the pathway is intrinsically diversity-oriented, the fact that 3 titres are lower than those of 1 and 2 ( Extended Data Table 2 ), rather argues that 3 arises from intermittent failures to suppress ACP 7 /cassette interactions. This phenomenon likely exemplifies the evolutionary challenges of achieving catalytic fidelity with acyl-ACP substrates which must interact with multiple partners, given the limited number of secondary structure and surface features offered by the small (ca. 10 kDa) domains 30 . This problem may be further aggravated by the evident structural plasticity of VirD ( Fig. 2ab ) and VirE which derives from helix a10, which could allow them to adapt to alternative partners. Furthermore, Module 7, which comprises only KS and ACP domains, is notably the sole PKS module in the Vir/Sna systems not to incorporate cis -acting modification domains which could kinetically outcompete b-methylation ( Fig. 1a ). In this context, we hypothesise that in addition to preferential recognition of ACP 5b , the atypical 3 domain composition of module 8 also plays a role in b-methylation programming. Notably, this module incorporates two copies of precisely the domains – heterocyclisation (HC) and peptidyl carrier protein (PCP) ( Fig. 1 ) – required for extension of the module 7 intermediate followed by oxazoline formation, suggesting that these domains kinetically and/or sterically outcompete the cassette enzymes, albeit imperfectly. It may be noteworthy that kinetic arguments are now also used to explain complex programming in iterative PKSs 33 and NRPS systems with trans -acting components 34 . Given that a high proportion of trans -AT PKSs systems comprise trans -acting enzymes including but not limited to b-branching cassettes 3,8 , it is likely that the existence of multiple control mechanisms is not limited to the virginiamycin system. We thus propose deblocking these latent chemistries as an innovative strategy for further diversifying polyketide structures. Declarations Online content Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org ... Acknowledgements We acknowledge financial support from the Agence Nationale de la Recherche (grant numbers ANR-11-JSV8-003-01, PKS-PPIs; ANR-16-CE92-0006-01, PKS STRUCTURE; and, ANR-20-CE93-0002-01, PKSOx to K.J.W.), the Université de Lorraine and the Centre National de la Recherche Scientifique (CNRS). We also acknowledge J. Davison for help with the molecular biology, Omar A. Rifi for assistance with production production and modification, and W. Shepard and M. Savko (Soleil Synchrotron, Proxima2) as well as J. Perez and A. Thureau (Soleil Synchrotron, Swing) for help with data acquisition. The NMR data were recorded on the NMR spectrometer of the Plateforme de Biophysique et Biologie Structurale (B2S) (IBSLor, UMS2008, CNRS-UL-INSERM). Analytical chemistry was performed on the Structural and Metabolomics Analyses Platform (PASM), SF4242, Université de Lorraine, EFABA, Vandœuvre‐lès‐Nancy, France. Author contributions A.G., K.J.W. and S.C. designed the study and carried out comparative sequence analysis. S.C. and B.C. designed and performed the molecular biology experiments. S.C. expressed and purified recombinant proteins, and S.C. and B.C. generated modified versions. S.C. performed the biophysical analyses, carried out the X-ray crystallography and SAXS analysis with A.G., and engineered the pathway inactivation with help from C.J. B.C. solved and analysed the ACP NMR structures. C.P. carried out the HPLC-MS experiments, and along with R.J.C., helped K.J.W. with data analysis and interpretation. All authors discussed the results. K.J.W., A.G. and S.C. wrote the manuscript, with input from BC. Competing interests The authors declare no competing interests. Additional information Extended data is available for this paper at https://doi.org/ Supplementary information . The online version contains supplementary material available at https://doi.org/ Correspondence and requests for materials should be addressed to B.C., K.J.W. or A.G. Reprints and permission information is available online at http://npg.nature.com/reprints References 1. Hertweck, C. The biosynthetic logic of polyketide diversity. Angew. Chem. Int. Ed. Engl. 48 , 4688–4716 (2009). 2. Weissman, K. J. Genetic engineering of modular PKSs: from combinatorial biosynthesis to synthetic biology. Nat. Prod. Rep. 33 , 203–230 (2016). 3. Helfrich, E. J. N. & Piel, J. Biosynthesis of polyketides by trans -AT polyketide synthases. Nat. Prod. Rep. 33 , 231–316 (2016). 4. Calderone, C. T. Isoprenoid-like alkylations in polyketide biosynthesis. Nat. Prod. 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(2022) doi:10.1039/d2np00007e. 34. Kaniusaite, M. et al. A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics. Chem. Sci. 10 , 9466–9482 (2019). Methods Bioinformatics analysis. trans -AT PKSs containing b-methylation modules were identified using refs. 3 and 8 . For comparative analysis of ACP domains, all PKS subunit sequences (with the exception of VirFG 10 ) were retrieved from the Protein data base (http://www.ncbi.nlm.nih.gov/protein), and domain boundaries were established relative to the solved structures of Vir ACPs 5a and 5b (PDB IDs: 2MF4, 4CA3) 10 . Sequence alignments shown in figures were generated using the NPS@ web server ( https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html ) 35 and the figures created with ESPript 36 . Materials and DNA manipulation. Biochemicals and media were purchased from VWR (glycerol, NaPi, NaCl, MgSO 4 ), BD (tryptone, yeast extract), Thermo Fischer Scientific (Tris, EDTA), Euromedex (isopropyl β-D-1-thiogalactopyranoside; IPTG), and Sigma-Aldrich (betaine, imidazole, Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), starch), and Roquette (corn steep). L-proline-2,5,5-D 3 and L-serine-2,3,3-D 3 were purchased from CDN Isotopes. The enzymes for genetic manipulation were purchased from Thermo Fisher Scientific. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin® Gel and PCR Clean‑up or NucleoSpin® Plasmid DNA kits (Macherey Nagel). Standard PCR reactions were performed with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific); and reactions were carried out on a Mastercycler Pro (Eppendorf). DNA sequencing was carried out by Eurofins. Strains and media. E. coli BL21(DE3) strains ( Supplementary Table 2 ) were obtained from Novagen and were cultured in LB medium (yeast extract 10 g L -1 , tryptone 5 g L -1 , NaCl 10 g L -1 , adjusted to pH 7.0 with NaOH) or on LB agar plates (LB medium supplemented with 20 g L -1 agar) at 37 °C. Streptomyces pristinaespiralis ATCC 25486 (DMSZ, Germany) and the derived mutants were sporulated on RP agar plates (20 g L -1 starch, 20 g L -1 soybean flour, 0.5 g L -1 valine, 0.5 g L -1 K 2 HPO 4, 1 g L -1 MgSO 4 × 7H 2 O, 2 g L -1 NaCl, 3 g L -1 CaCO 3 , 20 g L -1 agar in tap water) for 7 days at 30 °C. All strains were maintained in 20% ( v / v ) glycerol and stored at -80 °C. E. coli ET12567/pUZ8002 was used for conjugation and appropriate antibiotics were added to LB liquid and agar cultures at the following concentrations: ampicillin 100 mg L -1 , kanamycin 50 mg L -1 , apramycin 25 mg L -1 , chloramphenicol 25 mg L -1 and nalidixic acid 25 mg L -1 . For metabolite production by S. pristinaespiralis and its mutant ( Supplementary Table 2 ), 20 µL of spores were used to inoculate 25 mL inoculum medium (10 g L -1 corn steep powder, 15 g L -1 saccharose, 10 g L -1 (NH 4 ) 2 SO 4 , 1 g L -1 K 2 HPO 4 , 3 g L -1 NaCl, 0.2 g L -1 MgSO 4 × 7H 2 O, 1.25 g L -1 CaCO 3 in tap water, pH 6.9), followed by incubation at 30 °C and 180 rpm on rotary shaker for 72 h. Production medium (25 g L -1 soybean flour, 7.5 g L -1 starch, 22.5 g L -1 glucose, 3.5 g L -1 yeast extract, 0.5 g L -1 ZnSO 4 × 7H 2 O, 6 g L -1 CaCO 3 in tap water, pH 6.0) was inoculated with 2% of precultures, and incubated at 30 °C, 180 rpm on a rotary shaker for 96 h. To evaluate its effect, certain cultures were supplemented with 2% XAD-16 resin (Sigma-Aldrich). For feeding experiments, cultures were supplemented individually with L-proline-2,5,5-D 3 or L-serine-2,3,3-D 3 , or a combination of L-proline-2,5,5-D 3 and L-serine-2,3,3-D 3 , at 4, 24 and 48 h after incubation, in equal portions, to a final concentration of 3 mM. Gene cloning and site-directed mutagenesis. All protein-encoding constructs were amplified directly from Streptomyces virginiae genomic DNA using forward and reverse primers incorporating Bam HI and Hind III restriction sites, respectively ( Supplementary Table 1 ), and were ligated into the sites of vector pBG-102 for VirE and VirD and its mutant (VirD E128Q) or pLM-302 for VirC. Vector pBG-102 codes for a His 6 -SUMO tag and pLM-302 codes for a His 6 -maltose binding protein (MBP) tag (Centre for Structural Biology, Vanderbilt University). Following cleavage of the tags, the proteins incorporated a non-native N-terminal Gly-Pro-Gly-Ser sequence. The sequences of all constructs were verified by DNA sequencing prior to protein expression studies. Site-directed mutations were introduced into ACP 5a and VirD by PCR using mutagenic oligonucleotides ( Supplementary Table 1 ) and Phusion High-Fidelity polymerase, followed by digestion of the parental DNA by 1 μL of Dpn I Fast digest (Thermo Fischer Scientific). The presence of the correct mutations was confirmed by sequencing. Expression and purification of recombinant proteins ACP domains, VirC, VirC quadruple mutant (C114A/Q334A/R335A/R338A), VirD, VirD E128Q and VirE. All constructs were transformed into E. coli BL21(DE3) cells and grown at 37 °C in LB medium supplemented with 50 mg mL -1 kanamycin to an A 600 of 0.8, and then IPTG added to a final concentration of 0.5 mM. Following incubation at 20 °C for 18 h, the cells were harvested by centrifugation at 3000 g for 30 min at 4 °C, and cell pellets stored immediately at ─80 °C. Vir ACP 5a and ACP 5b purification was performed as described previously 10 , and all APC 5a mutants, ACP 5a ─ACP 5b didomain, ACP 6 and ACP 7 purified using the same method. In the case of all proteins of the b-methylation cassette, the cell pellets were suspended in His-buffer (50 mM NaPi pH 7.5, 250 mM NaCl, 10 % glycerol for VirC and the VirC quadruple mutant, or 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 10 % glycerol (VirD, VirD E128Q and VirE)) containing 8 U mL -1 of Benzonase (Merck) and 5 mM MgSO 4 . Cells were lysed by sonication and clarified by centrifugation (35,000 g for 40 min). Cell extracts were loaded onto a 5 ml HisTrap column (Cytiva) and washed with resuspension buffer supplemented with 20 mM imidazole. The supernatant was loaded onto a HisTrap 5 mL column equilibrated with His-buffer using an Akta Pure system (Cytiva). The proteins were eluted using a linear gradient of 0–50% His-elution buffer (50 mM NaPi pH 7.5, 250 mM NaCl, 300 mM imidazole for VirC and the VirC quadruple mutant or 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 300 mM imidazole (VirD, VirD E128Q and VirE)) over ten column volumes. All His 6 -tagged constructs were then incubated with His-tagged human rhinovirus 3C protease (1 µM) for 12─16 h at 4 °C to cleave the affinity/solubility tags. The constructs were then separated from the remaining His-tagged proteins by loading onto a HisTrap 5 mL column, followed by elution in resuspension buffer containing 20 mM imidazole. VirD, VirD E128Q and VirE were subsequently injected onto a Q-sepharose column (trimethylammonium on 6% agarose) equilibrated in buffer (20 mM Tris-HCl pH 8.5, 20 mM NaCl, 10 % glycerol). All proteins were then eluted using an NaCl gradient (100 mM─1 M) at 5 mL min −1 . Eluted fractions found to contain protein of the correct molecular weight as judged by SDS-PAGE analysis were pooled, concentrated using an Amicon Ultracel-10 (Merck Millipore) by centrifugation at 4000 g , and loaded onto a Superdex 200 16/60 (Cytiva) equilibrated with 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol (VirD, VirD E128Q and VirE) or a Superdex 75 16/60 column (Cytiva) (VirC and the VirC quadruple mutant). Following a concentration step, the purity of the recombinant proteins was determined by SDS-PAGE ( Extended Data Fig. 1 ), and their concentrations were determined by NanoDrop (or Qubit for ACP 6 ) (Thermo Scientific), with extinction coefficients calculated using the ExPASy ProtParam tool 37 . Expression of labelled protein samples for structural biology. Seleniated wild type VirD was produced in M9 minimal medium (50 mM Na 2 HPO4, 22 mM KH 2 PO4, 10 mM NaCl, 20 mM NH 4 Cl, adjusted to pH 7.2 with NaOH) for SAD/MAD phasing. Autoclaved M9 medium was supplemented with 50 mg L −1 of thiamine and riboflavin, 4 g L −1 glucose, 100 μM CaCl 2 , 2 mM MgSO 4 , 40 mg L −1 selenomethionine, and 40 mg L −1 of the 19 amino acids, based on the methionine biosynthesis inhibition method 38 . 13 C, 15 N-enriched Vir ACP 5a , ACP 6 and ACP 7 were produced in M9 medium supplemented with 15 NH 4 Cl (0.5 g L -1 ) and 13 C-glucose (2.0 g L -1 ), as the only sources of nitrogen and carbon. The labelled proteins were purified to homogeneity as described above. Svp-catalysed modification of ACP domains and verification by HPLC-MS. Following size exclusion chromatography, apo -ACPs (1 mM) were incubated in buffer (20 mM Tris-HCl pH 8.5) with 5 mM (acyl-)CoASH, 40 mM PPTase Svp 14 , 10 mM MgCl 2 and 50 mM TCEP for 22 h at 20 °C. The ACPs were then purified using a Superdex 75 16/60 column (Cytiva) equilibrated in 20 mM Tris-HCl pH 8.5, 250 mM NaCl, 50 mM TCEP. Quantitative modification was verified for all of the ACPs by HPLC-MS ( Extended Data Fig. 2 ) using either a Thermo Scientific Orbitrap ID-X Tribrid Mass Spectrometer, or an LTQXL mass spectrometer, both equipped with an in-line photodiode array detector (PDA) and an atmospheric pressure ionization interface operating in electrospray mode (ESI). All samples were diluted with Milli-Q water to a concentration of 50 µM and injected onto an Alltima™ C18 column (2.1 × 150 mm, 5 µm particle size). Analysis was carried out with Milli-Q water containing 0.1% TFA (A) and acetonitrile containing 0.1% TFA (B), using the elution profile: 0−15 min, linear gradient from 10−98% solvent B; 15−20 min, constant 98% solvent B; 20.1−26 min, constant 10% solvent B. In the case of the LTQXL, MS scans were performed in ESI + in the mass range m / z = 100−2000, at 3 K resolution, with MS parameters as follows: spray voltage, 5 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 20, 5 and 5 arbitrary units min -1 ; capillary temperature, 350 °C; capillary voltage, 7 V; tube lens, split lens and front lens voltages 180 V, ─22 V and ─11.75 V, respectively. MS data acquisition was carried out using the Xcalibur v. 2.1 software (Thermo Scientific). For the Orbitrap, MS scans were performed in heated ESI positive ion mode (HESI + ) in the mass range m / z = 150−2000, at 7.5 K or 60 K resolution (full width of the peak at its half maximum, fwhm, at m / z = 200) with MS parameters as follows: spray voltage, 4 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 30, 5 and 5 arbitrary units min -1 ; vaporiser and ion transfer tube temperatures were both set at 300 °C; maximum injection time, 50 ms; AGC target: 100000; normalised AGC target: 25%; microscans, 10; RF-lens, 35%; data type, profile. Mass spectrometer calibration was performed using the Pierce FlexMix calibration solution (Thermo Scientific). MS data acquisition was carried out using the Xcalibur v. 4.3 software (Thermo Scientific). For data obtained at low resolution (3 or 7.5 K), only the major isotopic peak was detected, while analysis at high resolution (60K) afforded the full isotopic spectrum ( Extended Data Fig. 2 ). Tryptophan fluorescence quenching. All tryptophan fluorescence spectroscopy experiments were performed on a SAFAS Fluorescence Xenius Spectrophotometer (SAFAS, France) in a 2 mL quartz cuvette. The excitation wavelength was fixed at 295 nm and emission spectra were collected between 300−400 nm with a slit width of 2 nm. The temperature was maintained at 25 °C by an external thermostatic water circulator. To measure protein-ligand interactions, recombinant VirC, VirD, VirD E128A mutant and VirE at 5 mM were allowed to equilibrate in TE buffer (20 mM Tris-HCl pH 8.5, 2 mM EDTA) for 10 min under constant stirring, before being titrated with ligand solutions. The proteins were analysed against increasing concentrations of ligand (0─150 mM), depending on the specific ligand used. Data from two independent experiments were analysed using nonlinear regression, with application of the one site-specific binding model (F = F max *X / (K d + X), where X is the ligand concentration, F is the fluorescence intensity, F max is the maximum specific binding and K d is the equilibrium binding constant) using SciDAVis v2.3.0. Circular dichroism measurements. Circular dichroism measurements were performed on a Chirascan CD (Applied Photophysics) in 100 mM NaPi, 150 mM NaF pH 8.0. Data were collected at 0.5 nm intervals in the wavelength range of 180─260 nm at 20 °C, using a temperature-controlled chamber. 30 mL of 100 mM ACP 5a , ACP5a E6761A/L6764N and VirD were analysed in a 0.01 cm cuvette, while 100 mL of 100 mM VirD E128Q was analysed in a 0.1 cm cuvette. Each spectrum represents the average of three scans, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone. Spectrum deconvolution was carried out using the CDNN2.1 software 39 ( Extended Data Fig. 1 ). Small-angle X-ray scattering (SAXS) data collection. SAXS data were acquired on the SWING beamline at the Synchrotron SOLEIL (France). The frames were recorded using an Eiger 4M detector at an energy of 12 keV. The distance between the sample and the detector was set to 2000 mm for VirD, VirE, holo -ACP 5b −VirC and holo -ACP 5b −VirE complexes, leading to scattering vectors q ranging from 0.0005−0.5 Å − 1 . The scattering vector is defined as 4p/l sinq, where 2q is the scattering angle. The protein samples were injected using the online automatic sample changer into a pre-equilibrated HPLC-coupled size-exclusion chromatography column (Bio-SEC 100 Å, Agilent), at a temperature of 15 °C. The eluted fractions were delivered using an online purification system developed on the SWING beamline 40 . After equilibrating the column in the protein buffer (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol), the buffer background was recorded (100 successive frames of 0.75 s). A 50 mL aliquot of the protein sample (at 5 mg mL -1 ) was then injected, and complete data sets were collected. The protein concentration downstream of the elution column was followed via the absorbance at 280 nm with an in situ spectrophotometer. In lieu of analysing several protein concentrations within a standard range ( e.g. , 0.1−10 mg mL − 1 ), the coupling of data collection to a gel filtration column allows analysis of multiple concentrations of protein within a single experiment, as many distinct positions within the elution peak are sampled during the course of the measurement (typically 50−100 frames are acquired) 40 . Following on from this, the dedicated in-house application FOXTROT was used to perform data reduction to absolute units, frame averaging, and solvent subtraction. Each acquisition frame of the experiment yielded a scattering spectrum, which was then analysed by FOXTROT to produce an R g (radius of gyration) as well as an I (0) value (the I (0) depends on the protein concentration at that position in the elution peak, as described by the Guinier law (approximation I ( q ) = I (0) exp(− q 2 R g 2 /3) for qR g < 1.3). Notably, observing a constant R g for a significant proportion of the concentrations present in the gel filtration peaks showed that the measurements were concentration-independent, and thus that they were effectively carried out under conditions of infinite dilution. Finally, all the frames exhibiting identical R g as a function of I (0) were corrected for buffer signal and averaged. This step ensured that the data reflected only the signal arising from the protein structure and not from intermolecular interactions. The distance distribution function P ( r ) and the maximum particle diameter D max were then calculated by Fourier inversion of the scattering intensity I ( q ) using GNOM 41 . The SAXS data are presented in Supplementary Table 3 . Molecular weights and oligomeric structures in solution from SAXS data. Classically, molecular weights can be derived from SAXS data using the I (0) and the known protein concentration. However, this method was not appropriate in our case, as the delay between exiting the gel filtration column and the SAXS data acquisition may have altered the concentrations. We therefore determined the molecular weights of the constructs using Bayesian Interference in PRIMUS 42 . SAXS data were recorded on wild type VirE, as well as VirC and VirE complexed with holo -ACP 5b . A model of a trimer of VirE was generated using ColabFold: AlphaFold2 19 . On the basis of the structural homology between the VirE model and the solved VirD crystal structure (r.m.s.d. 3.38 calculated based on 200 C a ), we generated a model of the holo -ACP 5b −VirE complex by superimposition on the crystal structure of holo -ACP 5b −VirD using PyMOL 43 . The quality of the 3D modelling was determined using CRYSOL 16 to compare the fit between the theoretical scattering curves from atomic coordinates with experimental scattering curves, and judged using the discrepancy c 2 , defined according to Konarev and colleagues 17 . SAXS data obtained on wild type VirC complexed with holo -ACP 5b were directly compared with that calculated 16 from the crystal structure of the acetyl-ACP D −CurD complex (PDB: 5KP6) 13 using CRYSOL 16 . Crystallisation and X-ray data collection. Se-VirD was purified and stored in buffer (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol) at a final concentration of 5 mg mL −1 . holo ACP 5b was stored in buffer (20 mM Tris-HCl pH 8.5, 250 mM NaCl, 50 mM TCEP) at a final concentration of 20 mg mL −1 . Prior to crystallization trials, sample homogeneity was checked by dynamic light scattering (DLS) using a Zetasizer NanoS (Malverne). Initial crystallisation hits were obtained using the Rigaku kit (Molecular Dimensions). The conditions consisted of 20% PEG 400, 20% PEG 800, 100 mM Tris-HCl, pH 7.5 for Se-VirD, while holo ACP 5b −Se-VirD crystallised in 100 mM chloride calcium, 30% PEG 1500, 10% 2-propanol, 100 mM imidazole-HCl, pH 6.5. Crystals grew in 10−15 days using the hanging drop method in Linbro® plates, with drops formed by mixing 2 mL of protein solution (ratio 1:4 for the holo -ACP 5b −Se-VirD complex, 5 mg mL −1 Se-VirD) with 1 mL of crystallisation buffer. Crystals were then soaked in crystallisation buffer containing 30% ethylene glycol prior to freezing in liquid nitrogen. X-ray diffraction data on Se-VirD and the holo ACP 5b −Se-VirD complex were collected at the SOLEIL synchrotron on the Proxima2 beamline. The crystals belong to the P4 1 2 1 2 and H3 space groups, respectively ( Supplementary Table 4 ). A complete MAD data set at four wavelengths was collected in order to solve the crystal structure of VirD. Data sets were indexed and integrated using XDS 44 and scaled by using pointless and aimless (CCP4 package). Structure determination and refinement. Initial phases were ultimately generated via SAD using the peak wavelength (λ = 0.979260 Å). Three high confidence Se sites were identified and refined by using the NCS using Phenix.autosol 45,46 . The figure of merit (FOM) from Phenix AutoSol is 0.32. Density modification and NCS were then used to improve the quality of the phases (FOM: 0.68 with a bias ratio of 1.36). The good quality of the electron density map allowed for building approximatively 80% of the backbone at 2.02 Å using Phenix.autobuild 47 . The final model of WT VirD was built using ARP/wARP 48 , followed by iterative cycles of manual rebuilding and refinement at 1.7 Å using COOT 49 and REFMAC5 50 . The structure of the holo -ACP 5b −VirD complex was solved by molecular replacement using a monomer of VirD as search model with the program MOLREP in CCP4 51,52 . The contrasted solution with final CC of 0.7252 and Tf/sig of 27.17, consists of 2 monomers of VirD in the asymmetric unit. The initial model was then refined by rigid body refinement at 3 Å followed by a restraint refinement at 2.1 Å resolution using REFMAC 5 CCP4 50 . The excellent quality of the electron density maps allowed us to locate two extra electron density in the F o F c map corresponding to two ACP 5b molecules in the asymmetric unit. The ACPs were then constructed manually in the electron density maps. Structure geometry was validated using the program MolProbity 53 . The structures of VirD and holo -ACP 5b −VirD contain 99.26% and 97.91% of the residues in the allowed region of the Ramachandran plot respectively and contain no outliers ( Supplementary Table 4 ). Figures were prepared using the program PyMOL 43 . Protein NMR data acquisition. All ACPs proteins samples were buffer exchanged via gel filtration into 100 mM sodium phosphate (pH 6.0), 1 mM EDTA and 1 mM TCEP, concentrated to 1 mM, and then 350 μL of the samples (including 10% D 2 O) were loaded into 4 mm NMR tubes. All NMR data were recorded at 25 °C on a Bruker DRX600 spectrometer equipped with a cryogenic probe (Unité Mixte de Service (UMS) 2008 Ingénierie-Biologie-Santé en Lorraine (IBSLor)). Backbone and sequential resonance assignments were obtained by the combined use of 2D 15 N− 1 H and 13 C− 1 H HSQC spectra and 3D HNCA, HNCACB, CBCA(CO)NH, HNHA, HBHA(CO)NH, HN(CA)CO, and HNCO experiments. Assignments of aliphatic side chain resonances were based on 2D aromatic 13 C− 1 H HSQC, (HB)CB(CGCDCE)HE, (HB)CB(CGCD)HD and 3D (H)CC(CO)NH, H(CC)(CO)NH, CCH−TOCSY, and HCCH-TOCSY experiments (reviewed in 54 ). To collect NOE-based distance restraints for the structure calculations, 3D 15 N NOESY-HSQC and 13 C NOESY-HSQC were recorded on uniformly 13 C, 15 N enriched samples using a mixing time of 120 ms. NMR data were processed using Topspin 3.2 (Bruker) and were analysed using NMRFAM-SPARKY 55 . Protein NMR structure calculations. Initial structures were generated using CYANA 3.98 software 56 . Starting from a set of manually-assigned NOEs, the standard CYANA protocol of seven iterative cycles of calculations was performed with NOE assignment by the embedded CANDID routine combined with torsion angle dynamics structure calculation 57 . In each cycle, 100 structures starting from random torsion angle values were calculated with 15,000 steps of torsion angle dynamics-driven simulated annealing. A total of 1822, 1208 and 1763 NOE-based distances, 110, 92 and 94 backbone angle restraints were used for structure calculation of the holo -ACP 5a , holo- ACP 6 and holo -ACP 7 domains, respectively ( Supplementary Table 5 ). The angle restraints were obtained from 13 Cα, 13 Cβ, 13 C′, 15 N, 1 HN, and 1 Hα chemical shifts using TALOS-N 58 with an assigned minimum range of ±20°. 4¢-phosphopantetheine-serine was created as a serine modified residue within the CYANA library using 4¢-phosphopantetheine coordinates from the solution structure of holo -ACP PfACP from Plasmodium falciparum (PDB ID: 2FQ0) 59 . The second stage consisted of the refinement of the 50 lowest CYANA target function conformers by restrained molecular dynamic (rMD) simulations in Amber 14 60,61 , following published protocols 62 . Phosphopantetheinyl serine library and force field parameters 63 were used for AMBER minimisation. The final representative ensembles correspond to the 20 conformers from each calculation with the lowest restraint energy terms. The structures of holo -ACP 5a , holo -ACP 6 and holo -ACP 7 contain 98.6%, 94.4% and 97.1% in the most favoured region and 1.4 %, 5.6% and 2.9% of the residues (non-glycine and non-proline) in the additional allowed region of the Ramachandran plot, respectively. PROCHECK statistics were calculated using PROCHECK-NMR 64 . The proportion of residues in the most favoured/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot for the ACPs are as follows: holo -ACP 5a (97.1/2.9/0/0); holo -ACP ­6 (94.3/5.7/0/0); holo -ACP 7 (92.4/7.1/0.1/0.4). Generation of S. pristinaespiralis pathway inactivation mutant. For construction of the pathway mutant, the pCRISPomyces-2 plasmid 25 was used for CRISPR-Cas9-based genome editing. Spacer sequences ( Supplementary Table 1 ) were chosen using the online CRISPy-web software 65 , and were generated by annealing two 24 nt oligonucleotides. Next, 1 kb homologous arms HAL and HAR were amplified by PCR, the pCRISPomyces-2 plasmid was linearised with the restriction enzyme Xba I (Thermo Fisher Scientific), and then assembly of the editing templates and the pCRISPomyces-2 plasmid was performed using the In-Fusion HD Cloning kit (Ozyme, France). Correct plasmid assembly was confirmed by diagnostic digestion and sequencing ( Supplementary Fig. 5 ). Recombinant plasmids were introduced into E. coli 12567 (pUZ8002) by electroporation. Conjugation of plasmids into Streptomyces spores was performed using the protocol described elsewhere 66 . Following conjugation, clearance of the plasmid was accomplished by repeated high-temperature cultivation (37 °C) for 2–3 days, followed by replica plating on selective and nonselective plates to confirm restoration of apramycin sensitivity. Apramycin-sensitive colonies were then picked into liquid ISP2 medium (4 g L -1 yeast extract, 4 g L -1 dextrose, 10 g L -1 malt extract adjusted to pH 7.3 with NaOH) for genomic DNA isolation using the Wizard Genomic DNA Purification Kit (Promega). Genomic modifications were confirmed by PCR and sequencing of the modified regions ( Supplementary Fig. 5 ). Analysis by HPLC-MS of the S. pristinaespiralis pathway inactivation mutant. S. pristinaespiralis cultures were extracted twice with ethyl acetate ( v / v ). When present, XAD-16 resin was harvested by sieving, and also extracted twice with ethyl acetate ( v / v ). The solvent was removed by evaporation, the extracts resuspended in 1:1 ACN/water ( v / v ) and then the sample was passed through a 0.4 µm syringe filter. HPLC-MS analysis was performed in positive and/or negative electrospray mode (ESI+/−) on the Thermo Scientific Orbitrap ID-X Tribrid Mass Spectrometer using an Alltima™ C18 column (2.1 × 150mm, 5 µm particle size) at 25°C (flow rate, 0.2 mL min -1 ). Separation was carried out with Milli-Q water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B), using the following elution profile: 0−48 min, linear gradient 5−95% solvent B; 48−54 min, constant 95% solvent B; 54−60 min, constant 5% solvent B. Mass spectrometry operating parameters were as described previously. Metabolite yields ( Extended Data Table 2 ) were estimated by generating a calibration curve using commercially-available virginiamycin M 1 (Sigma-Aldrich), over the concentration range of 0.00128−20 mg L -1 (10 mL of each sample was injected). This approach afforded a linear correlation between the quantity of metabolite and the respective integrated peak area in the extracted ion chromatogram (EIC) (the areas of the peaks corresponding to the parental ions [M+H] + were used systematically) ( Supplementary Fig. 4 ). For analysis of metabolite yields in extracts, following conversion of peak areas to titres, the results were divided by 200 to correct for the enrichment of the sample during preparation, as the extracts from 20 mL of culture were resuspended in 100 mL of solvent prior to HPLC-MS analysis (as with the standard, 10 mL of each sample was injected). Data availability Crystal structures of VirD and the holo -ACP 5b −VirD complex have been deposited in the Protein Data Bank with their respective diffraction data under accession codes 8AHZ and 8AHQ, respectively. Coordinates and chemical shifts for the NMR structures of holo -ACP 5a , holo -ACP 6 and holo -ACP 7 has been deposited in the Biological Magnetic Resonance Bank with accession codes 8A7Z, 8AIG, and 8ALL, respectively. SAXS and HPLC-MS data have been deposited in the data repository DOREL (Données de la Recherche Lorraines) [ https://doi-org.insis.bib.cnrs.fr/10.12763/PEYXHP ]. The remaining data supporting this study are included in the Supplementary Information. Source data are provided with this paper, and all biological materials are available from the authors upon request. References 35. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 , 4673–4680 (1994). 36. Gouet, P., Robert, X. & Courcelle, E. 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PREFACE. in Protein NMR Spectroscopy (Second Edition) (eds. Cavanagh, J., Fairbrother, W. J., Palmer, A. G., Rance, M. & Skelton, N. J.) v–vi (Academic Press, 2007). 55. Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31 , 1325–1327 (2015). 56. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278 , 353–378 (2004). 57. Herrmann, T., Güntert, P. & Wüthrich, K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319 , 209–227 (2002). 58. Shen, Y. & Bax, A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 56 , 227–241 (2013). 59. Sharma, A. K., Sharma, S. K., Surolia, A., Surolia, N. & Sarma, S. P. 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E., Weber, T. & Lee, S. Y. CRISPy-web: An online resource to design sgRNAs for CRISPR applications. Synth. Syst. Biotechnol. 1 , 118–121 (2016). 66. Hopwood, D.A., Kieser, T., Bibb, M., Buttner, M. & Chater, K. Practical Streptomyces Genetics . (John Innes Foundation, 2000). 67. Walker, P. D., Weir, A. N. M., Willis, C. L. & Crump, M. P. Polyketide β-branching: diversity, mechanism and selectivity. Nat. Prod. Rep. 38 , 723–756 (2021). 68. Pan, G. et al. Discovery of the leinamycin family of natural products by mining actinobacterial genomes. Proc. Natl. Acad. Sci. U. S. A. 114 , E11131–E11140 (2017). 69. Erol, O. et al. Biosynthesis of the myxobacterial antibiotic corallopyronin A. ChemBioChem 11 , 1253–1265 (2010). 70. Sucipto, H., Wenzel, S. C. & Müller, R. Exploring chemical diversity of α-pyrone antibiotics: molecular basis of myxopyronin biosynthesis. ChemBioChem 14 , 1581–1589 (2013). 71. Tang, G.-L., Cheng, Y.-Q. & Shen, B. Leinamycin biosynthesis revealing unprecedented architectural complexity for a hybrid polyketide synthase and nonribosomal peptide synthetase. Chem. Biol. 11 , 33–45 (2004). 72. Helfrich, E. J. N. et al. Automated structure prediction of trans -acyltransferase polyketide synthase products. Nat. Chem. Biol. 15 , 813–821 (2019). 73. Jin, Q., Jin, Z., Zhang, L., Yao, S. & Li, F. Probing the molecular mechanisms for pristinamycin yield enhancement in Streptomyces pristinaespiralis . Curr. Microbiol. 65 , 792–798 (2012). Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedData.docx Collinetal.SI.docx Cite Share Download PDF Status: Published Journal Publication published 10 Mar, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2103032","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":144253057,"identity":"e173dfe7-a1ce-4389-a8bf-b38228195a57","order_by":0,"name":"Sabrina Collin","email":"","orcid":"","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"","lastName":"Collin","suffix":""},{"id":144253058,"identity":"ce816fce-30ec-46b1-9ee1-472bb2ce8418","order_by":1,"name":"Russell Cox","email":"","orcid":"https://orcid.org/0000-0002-1844-0157","institution":"University of Hannover","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Russell","middleName":"","lastName":"Cox","suffix":""},{"id":144253059,"identity":"d13d6eea-99d8-4d19-943d-91d0d1bfccd5","order_by":2,"name":"Cédric Paris","email":"","orcid":"","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cédric","middleName":"","lastName":"Paris","suffix":""},{"id":144253060,"identity":"fe2f2ec7-cbb6-4b6a-be18-8e4dd25f5d15","order_by":3,"name":"Christophe Jacob","email":"","orcid":"https://orcid.org/0000-0001-8522-2865","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Jacob","suffix":""},{"id":144253061,"identity":"c67fcfdf-46a5-4806-91dd-e5c17197b774","order_by":4,"name":"Benjamin Chagot","email":"","orcid":"https://orcid.org/0000-0002-9153-0060","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Chagot","suffix":""},{"id":144253062,"identity":"0f7249bc-ecf7-486a-a31a-054dd28a505a","order_by":5,"name":"Kira Weissman","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3012-2960","institution":"University of Lorraine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kira","middleName":"","lastName":"Weissman","suffix":""},{"id":144253063,"identity":"c9b1b557-377c-404f-b676-0f8f11ec859a","order_by":6,"name":"Arnaud Gruez","email":"","orcid":"","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Arnaud","middleName":"","lastName":"Gruez","suffix":""}],"badges":[],"createdAt":"2022-09-26 05:45:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2103032/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2103032/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-023-36974-3","type":"published","date":"2023-03-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27864241,"identity":"e294da75-6afc-4b1f-8dc2-a8a000cb0eff","added_by":"auto","created_at":"2022-10-17 14:11:16","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":816625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiosynthesis of metabolites \u003c/strong\u003e1\u003cstrong\u003e−\u003c/strong\u003e3 \u003cstrong\u003ein \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptomyces\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u0026nbsp;a.\u003c/strong\u003e Genetic and protein organisation of the biosynthetic pathway to virginiamycin M \u003cstrong\u003e1\u003c/strong\u003e in \u003cem\u003eStreptomyces virginiae\u003c/em\u003e (Vir)\u003csup\u003e9\u003c/sup\u003e and \u003cem\u003eStreptomyces pristinaespiralis\u003c/em\u003e (Sna)\u003csup\u003e26\u003c/sup\u003e. The D-stereochemistry of the Pro is assumed based on the presence of an epimerisation (E) domain in module 10. The enzymes responsible for the dehydrogenation in module 8 to yield the thiazole and the post-assembly line proline dehydrogenation to afford the 2-pyrroline moiety, have not been conclusively identified. Metabolite \u003cstrong\u003e2\u003c/strong\u003e, the direct precursor of \u003cstrong\u003e1\u003c/strong\u003e, lacks the proline dehydrogenation. The functional domains within the subunits are shown as spheres. Key to the domains/enzymes: BCDH, branched-chain a-keto acid dehydrogenase; ACP, acyl carrier protein; KS, ketosynthase; DH, dehydratase (° indicates an inactive domain); KR, ketoreductase; \u003cem\u003eC\u003c/em\u003e-MT, \u003cem\u003eC\u003c/em\u003e-methyl transferase; C, condensation; A, adenylation; PCP, peptidyl carrier protein; HC, heterocyclisation; E, epimerisation; TE, thioesterase; AT, acyl transferase; TE\u003csub\u003eII\u003c/sub\u003e, proof-reading thioesterase; PPTase, phosphopantetheinyl transferase. \u003cstrong\u003eb.\u003c/strong\u003e Series of transformations catalysed by the b-methylation cassette (both the Vir and Sna homologues are indicated), in which acetate generated by decarboxylation of malonate tethered to the ACP donor (ACP\u003csub\u003eD\u003c/sub\u003e), is initially condensed with the polyketide intermediate attached to the ACP acceptor (ACP\u003csub\u003eA\u003c/sub\u003e). Metabolites \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e2\u003c/strong\u003e arise from b-methylation in module 5, which our data show occurs preferentially on ACP\u003csub\u003e5b\u003c/sub\u003e. \u003cstrong\u003ec. \u003c/strong\u003eProposed pathway leading to doubly methylated (blue dots) derivative \u003cstrong\u003e3\u003c/strong\u003e, in which b-methylation occurs additionally following chain extension in module 7. The proposed structure of \u003cstrong\u003e3\u003c/strong\u003e is based on its exact mass and the results of feeding isotopically-labelled amino acids (\u003cstrong\u003eExtended Data Fig.\u003c/strong\u003e \u003cstrong\u003e8\u003c/strong\u003e). The configuration of the second b-branch in \u003cstrong\u003e3\u003c/strong\u003e has been extrapolated from the known stereochemistry of b-branching\u003csup\u003e67\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/977715195599aa10d8a118c7.jpeg"},{"id":27864741,"identity":"af9418d9-4126-4e41-a499-edb788a34856","added_by":"auto","created_at":"2022-10-17 14:16:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1487322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure analysis of the VirD/ACP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e5b\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e interaction, and basis for specificity. a.\u003c/strong\u003e VirD crystal structure (PDB ID: 8AHZ). The three monomers of VirD are shown in cartoon representation and the three polypeptide chains are coloured in white, teal, and light blue. Helix a10, which is partially defined in the electron density maps, is indicated in marine blue. \u003cstrong\u003eb.\u003c/strong\u003e \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e−VirD crystal structure (PDB ID: 8AHQ), colour-coded as in \u003cstrong\u003ea. \u003c/strong\u003eand with ACP\u003csub\u003e5b\u003c/sub\u003e shown in lime green. Within the context of the complex, helix a10 is fully-structured. The side chains of S6871 and the Ppant arm, only the proximal end of which is visible in the electron density, are shown in stick representation (oxygen atoms are indicated in red, nitrogen atoms in blue, carbon atoms in white, and the sulfur atom in yellow). \u003cstrong\u003ec.\u003c/strong\u003e Zoom into the VirD active site. The side and main chains of N6865, L6869, L6894 and D6870 are shown in ball-and-stick representation. The hydrogen bond network between the residues and water molecules (red spheres) are represented as dashed lines. The positively-charged arginines of VirD (R125 and R192) participate in salt bridges with the phosphate moiety and D6870 of the ACP\u003csub\u003e5b\u003c/sub\u003e. The omit map of the Ppant arm and the S6871 is contoured at 2s in white and at 3s in red. The distance of 13.9 Å between S6871 and the VirD catalytic E128 is shown as a red dashed line. The oxyanion hole established by the N-terminal portion of helix a3 comprises the NH moiety of D6870, I6872 and L6873. The orientation of the side chain of S6871 is maintained by the oxyanion hole as well as the side chain orientation of D6870.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/69ee5c9d725c4277c7eda334.png"},{"id":27864042,"identity":"0d141c9f-8953-497d-bc1e-c14c89dc9a9f","added_by":"auto","created_at":"2022-10-17 14:06:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":759330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuperimposition of a selection of ACPs on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eholo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-ACP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e5b\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. a. \u003c/strong\u003eSuperimposition of the average NMR structures of virginiamycin \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e (in deep blue), \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e6\u003c/sub\u003e (in white) and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e (in firebrick red) on the crystal structure of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e (in lime green). a-helices are shown in cartoon representation and the Ppant cofactor as sticks. \u003cstrong\u003eb.\u003c/strong\u003e Superimposition of the NMR structures of Mup ACP\u003csub\u003e3a \u003c/sub\u003e(in marine blue) and ACP\u003csub\u003e3b\u003c/sub\u003e (in orange) (PDB ID: 2L22) on the crystal structure of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e (in lime green) (PDB ID: 8AHQ), reveals an r.m.s.d. of 0.949 Å (67 Ca) and 1.106 Å (59 Ca), respectively, and no substantial deviation in terms of the helix a3 orientation. The tryptophan flags of Mup ACP\u003csub\u003e3a \u003c/sub\u003eand ACP\u003csub\u003e3b\u003c/sub\u003e\u003csup\u003e7\u003c/sup\u003e and the corresponding phenylalanine of Vir ACP\u003csub\u003e5b\u003c/sub\u003e, are shown in ball-and-stick representation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/16b7456461a1e5592365d428.png"},{"id":27864038,"identity":"43025b68-7f25-4de5-a7b4-e7daa99d3b78","added_by":"auto","created_at":"2022-10-17 14:06:16","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":401430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eholo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-ACP surface features at the observed interaction interface with VirD. \u003c/strong\u003eSurface representation of the \u003cem\u003eholo\u003c/em\u003e-ACPs: \u003cstrong\u003ea.\u003c/strong\u003e ACP\u003csub\u003e5b\u003c/sub\u003e (PDB ID: 8AHQ); \u003cstrong\u003eb.\u003c/strong\u003e ACP\u003csub\u003e7\u003c/sub\u003e (PDB ID: 8ALL); \u003cstrong\u003ec.\u003c/strong\u003e ACP\u003csub\u003e5a \u003c/sub\u003e(PDB ID: 8A7Z); and, \u003cstrong\u003ed.\u003c/strong\u003e ACP\u003csub\u003e6 \u003c/sub\u003e(PDB ID: 8AIG). The amino acids are coloured according to their properties: positively-charged residues (R or K) in light blue, histidine in navy blue, negatively-charged residues (D or E) in red, polar residues (T, S, N) in green, and hydrophobic residues in white. The Ppant prosthetic group is shown in sphere representation with the oxygen atoms in red, nitrogen atoms in blue, carbon atoms in white, and the sulfur atom in yellow.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/30c3f5ad0b632b0d56b65e9b.jpeg"},{"id":27864044,"identity":"168b0def-15b0-4a81-b079-5a7620a03bf3","added_by":"auto","created_at":"2022-10-17 14:06:17","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":254200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMS analysis of metabolites\u003c/strong\u003e 1\u003cstrong\u003e−\u003c/strong\u003e3. \u003cstrong\u003ea.\u003c/strong\u003e Extracted ion chromatograms (EICs) based on the calculated accurate masses for metabolites \u003cstrong\u003e1\u003c/strong\u003e−\u003cstrong\u003e3\u003c/strong\u003e. In each case, the retention time and integrated peak areas (peaks defined by the blue lines) are indicated. \u003cstrong\u003eb.\u003c/strong\u003e Accurate mass determination of metabolites \u003cstrong\u003e1\u003c/strong\u003e−\u003cstrong\u003e3\u003c/strong\u003e, indicating the calculated and observed masses (Z = 1), and the mass errors.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/96efa462990a1ab9d680ce8e.jpeg"},{"id":34098724,"identity":"9d1b150b-a782-484b-8c81-78216ab1f4c9","added_by":"auto","created_at":"2023-03-11 08:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3191965,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/4a61e1eb-4982-4cf4-abfd-bbce6be5885a.pdf"},{"id":27864242,"identity":"bf6ec7fb-a8f4-4698-9920-9d4b01d31d5b","added_by":"auto","created_at":"2022-10-17 14:11:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6947052,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/9ea927845b0f793455d04428.docx"},{"id":27864040,"identity":"95a68303-e05b-4eef-a3c5-819af6d3e0ef","added_by":"auto","created_at":"2022-10-17 14:06:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1994924,"visible":true,"origin":"","legend":"","description":"","filename":"Collinetal.SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-2103032/v1/bc41a772bb506eda204356df.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Decrypting the programming of β-methylation in virginiamycin M biosynthesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNature deploys an assembly line strategy to construct polyketide specialised metabolites, in which each task is assigned to a specific enzyme. In the prototypical \u003cem\u003ecis\u003c/em\u003e-AT systems, the majority of these functions are present within catalytic domains of gigantic multienzymes called polyketide synthases (PKSs)\u003csup\u003e1\u003c/sup\u003e. The functional domains are clustered into modules, where each module is typically responsible for carrying out one round of chain extension and\u0026nbsp;b-processing of the resulting intermediate. In addition to the three domains which are essential to chain building (acyl transferase (AT), ketosynthase (KS), and acyl carrier protein (ACP)), many modules also harbour optional domains which modify the oxidation state of the\u0026nbsp;b-keto group resulting from the condensation reaction. The division-of-labour organisation of PKS systems makes them attractive targets for synthetic biology approaches aiming at generating high-value derivatives\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRelative to the \u003cem\u003ecis\u003c/em\u003e-AT PKSs, the \u003cem\u003etrans\u003c/em\u003e-AT systems\u003csup\u003e3\u003c/sup\u003e (\u003cstrong\u003eFig. 1a\u003c/strong\u003e) incorporate\u0026nbsp;one or more free-standing enzyme activities and a wider variety of enzymatic functions, including cassettes of enzymes which introduce\u0026nbsp;b-branching into the polyketide intermediates\u003csup\u003e4\u003c/sup\u003e. A common modification is\u0026nbsp;b-methylation, which involves five discrete proteins (\u003cstrong\u003eFig. 1b\u003c/strong\u003e): (i) a malonate-loaded ACP (called ACP donor, ACP\u003csub\u003eD\u003c/sub\u003e); (ii) a condensation-inactive KS domain (KS\u003csup\u003e0\u003c/sup\u003e) which generates acetyl-ACP\u003csub\u003eD\u003c/sub\u003e from the malonyl-ACP; (iii) a 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) homologue which catalyses attack of the acetate-derived nucleophile on the\u0026nbsp;b-keto group of the polyketide chain attached to an acceptor ACP (ACP\u003csub\u003eA\u003c/sub\u003e), yielding an HMG-\u003cem\u003eS\u003c/em\u003e-ACP\u003csub\u003eA\u003c/sub\u003e thioester; (iv) an enoyl-CoA hydratase (ECH) homolog (ECH\u003csub\u003e1\u003c/sub\u003e) that serves as a dehydratase to produce the corresponding\u0026nbsp;a,b-unsaturated thioester; and finally, (v) a second ECH homolog (ECH\u003csub\u003e2\u003c/sub\u003e) that catalyses decarboxylation to afford the\u0026nbsp;b-methyl product. Variation of the electrophile and nucleophile structures, and/or HMG processing sequences, gives access to further types of\u0026nbsp;b-functionality\u003csup\u003e4,5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAn intriguing feature of\u0026nbsp;b-modification is how the system selects which polyketide-ACP\u003csub\u003eA\u003c/sub\u003e intermediate to target, as every round of chain extension yields a potential\u0026nbsp;b-keto substrate. In principle, gate-keeping by the HMGS would be sufficient to direct the whole cassette, as no downstream enzymes can act in the absence of this chemistry\u003csup\u003e6\u003c/sup\u003e. Previous work identified a sequence motif including a conserved Trp flag characteristic of ACP domains in modules targeted for\u0026nbsp;b-methylation\u003csup\u003e7\u003c/sup\u003e. As the majority of the residues are confined to the domain core, a model was proposed\u003csup\u003e7,8\u003c/sup\u003e in which burial of the Trp side chain gouverns the orientation of helices\u0026nbsp;a2 and\u0026nbsp;a3 within the ACP four\u0026nbsp;a-helix bundle, allowing both specific residues on helix\u0026nbsp;a3 and the substrate to interact with the HMGS. Nonetheless, the detailed recognition mechanism remains obscure, hampering efforts to install\u0026nbsp;b-branches at will by redirecting HMGS cassettes to alternative ACP\u003csub\u003eA\u003c/sub\u003e-bound intermediates.\u003c/p\u003e\n\u003cp\u003eHere we aimed to understand the control of\u0026nbsp;b-methylation during biosynthesis of virginiamycin (Vir) M \u003cstrong\u003e1\u003c/strong\u003e by a hybrid \u003cem\u003etrans\u003c/em\u003e-AT PKS-nonribosomal peptide synthetase (NRPS) system\u003csup\u003e9\u003c/sup\u003e (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). In this case,\u0026nbsp;b-modification was thought to occur exclusively during chain extension by module 5 (M5) (\u003cstrong\u003eFig. 1ab\u003c/strong\u003e)\u003csup\u003e3\u003c/sup\u003e. As is typical of\u0026nbsp;b-branching modules\u003csup\u003e8\u003c/sup\u003e, Vir M5 incorporates a tandem of ACP\u003csub\u003eA\u003c/sub\u003e domains, ACP\u003csub\u003e5a\u003c/sub\u003e and ACP\u003csub\u003e5b\u003c/sub\u003e. We show by Trp fluorescence quenching that the Vir HMGS, ECH\u003csub\u003e1\u003c/sub\u003e and ECH\u003csub\u003e2\u003c/sub\u003e homologues (VirC\u0026minus;VirE) preferentially bind ACP\u003csub\u003e5b\u003c/sub\u003e in its \u003cem\u003eholo\u003c/em\u003e and substrate mimic forms, consistent with in-series function in which ACP\u003csub\u003e5a\u003c/sub\u003e participates in chain extension and ACP\u003csub\u003e5b\u003c/sub\u003e in\u0026nbsp;b-methylation\u003csup\u003e10\u003c/sup\u003e. Comparative structural analysis of multiple Vir and mupirocin ACPs at high-resolution reveals essentially identical folds, excluding helix\u0026nbsp;a3 orientation as the basis for specific recognition. Instead, the crystal structure of the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD complex identifies an ACP interaction motif centred on the phophopantetheine (Ppant) prosthetic arm and surrounding secondary structural elements, with specificity conferred via distinctive electrostatic surface features of the domain combined with precise Ppant positioning. We also show \u003cem\u003ein vitro\u003c/em\u003e that Vir \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e is recognised by the cassette enzymes, and identify a doubly\u0026nbsp;b-methylated Vir M derivative in production extracts. The ca. 150-fold lower titres of the analogue relative to Vir M \u003cstrong\u003e1\u003c/strong\u003e imply that this second\u0026nbsp;b-methylation is suppressed \u003cem\u003ein vivo\u003c/em\u003e. Taken together, our data show that b-methylation programming relies on at least two distinct control mechanisms, but remains imperfect, identifying the deactivation of such measures as a promising strategy for polyketide analoguing.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eBinding of VirC\u0026minus;E to Vir ACPs.\u0026nbsp;\u003c/strong\u003eWe first assessed the interaction between recombinant \u003cem\u003eapo\u003c/em\u003e- and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e and ACP\u003csub\u003e5b\u003c/sub\u003e and VirC\u0026minus;VirE from the Vir pathway of \u003cem\u003eStreptomyces virginiae\u003c/em\u003e\u003csup\u003e9\u003c/sup\u003e, with ACPs 6 and 7 from non\u0026nbsp;b-methylation modules selected as controls \u003cem\u003ein vitro\u003c/em\u003e (\u003cstrong\u003eExtended Data Figs. 1\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e2\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Supplementary Tables 1\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e). While the \u003cem\u003eapo\u003c/em\u003e forms are not physiologically relevant, the \u003cem\u003eholo\u003c/em\u003e proteins are present at several stages of the catalytic cycles\u003csup\u003e11\u003c/sup\u003e. No binding was detected using tryptophan fluorescence quenching\u003csup\u003e12\u003c/sup\u003e between VirC\u0026minus;VirE\u0026nbsp;and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e, although \u003cem\u003eapo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e was weakly bound (K\u003csub\u003ed\u003c/sub\u003e = 166\u0026nbsp;\u0026plusmn;\u0026nbsp;18\u0026nbsp;mM, 102\u0026nbsp;\u0026plusmn;\u0026nbsp;14\u0026nbsp;mM and 653\u0026nbsp;\u0026plusmn;\u0026nbsp;171\u0026nbsp;mM,\u0026nbsp;respectively)\u0026nbsp;(\u003cstrong\u003eExtended Data Table 1, Supplementary Figs. 1\u003c/strong\u003e\u0026minus;\u003cstrong\u003e3\u003c/strong\u003e). While interaction with \u003cem\u003eapo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e was similarly weak (77\u0026nbsp;\u0026plusmn;\u0026nbsp;10\u0026nbsp;mM, 178\u0026nbsp;\u0026plusmn;\u0026nbsp;26\u0026nbsp;mM and 171\u0026nbsp;\u0026plusmn;\u0026nbsp;31\u0026nbsp;mM), the three enzymes showed good affinity towards \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e (3.8\u0026nbsp;\u0026plusmn;\u0026nbsp;0.5\u0026nbsp;mM, 2.9\u0026nbsp;\u0026plusmn;\u0026nbsp;0.5\u0026nbsp;mM and 6.8\u0026nbsp;\u0026plusmn;\u0026nbsp;1.5\u0026nbsp;mM), consistent with\u0026nbsp;an important role in recognition for the Ppant arm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo provide a more native context to these assays, we also analysed binding of VirC\u0026minus;VirE\u0026nbsp;to the \u003cem\u003eholo\u003c/em\u003e-ACP\u0026shy;\u003csub\u003e5a\u003c/sub\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e didomain\u0026nbsp;(\u003cstrong\u003eExtended Data Figs. 1\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e2\u003c/strong\u003e). The observed binding affinities (K\u003csub\u003ed\u003c/sub\u003e = 17\u0026nbsp;\u0026plusmn;\u0026nbsp;2\u0026nbsp;mM, 19\u0026nbsp;\u0026plusmn;\u0026nbsp;3\u0026nbsp;mM and\u0026nbsp;42\u0026nbsp;\u0026plusmn;\u0026nbsp;10\u0026nbsp;mM) (\u003cstrong\u003eExtended Data Table 1, Supplementary Figs. 1\u003c/strong\u003e\u0026minus;\u003cstrong\u003e3\u003c/strong\u003e), are within the same order of magnitude as for the discrete \u003cem\u003eholo\u003c/em\u003e domains, and thus we find no evidence for cooperative binding of tandem ACP\u003csub\u003eA\u003c/sub\u003es as previously proposed\u003csup\u003e13\u003c/sup\u003e. Furthermore, no binding was detected by VirC\u0026minus;VirE to control ACP\u003csub\u003e6\u003c/sub\u003e in either its \u003cem\u003eapo\u003c/em\u003e or \u003cem\u003eholo\u003c/em\u003e forms. However, unexpectedly, both \u003cem\u003eapo\u003c/em\u003e- and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e behaved similarly to the analogous forms of ACP\u003csub\u003e5b\u003c/sub\u003e with\u0026nbsp;VirC\u0026minus;VirE\u0026nbsp;(\u003cem\u003eapo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e: 75\u0026nbsp;\u0026plusmn;\u0026nbsp;7\u0026nbsp;mM, 75\u0026nbsp;\u0026plusmn;\u0026nbsp;7\u0026nbsp;mM and 301\u0026nbsp;\u0026plusmn;\u0026nbsp;126\u0026nbsp;mM; \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e: 18\u0026nbsp;\u0026plusmn;\u0026nbsp;3\u0026nbsp;mM, 4.3\u0026nbsp;\u0026plusmn;\u0026nbsp;0.2\u0026nbsp;mM and 22\u0026nbsp;\u0026plusmn;\u0026nbsp;5\u0026nbsp;mM) (\u003cstrong\u003eExtended Data Table 1, Supplementary Figs. 1\u003c/strong\u003e\u0026minus;\u003cstrong\u003e3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFinally, we evaluated binding of VirC\u0026minus;VirE to ACP\u003csub\u003e5a\u003c/sub\u003e and ACP\u003csub\u003e5b\u003c/sub\u003e modified, albeit imperfectly, to mimic the native substrates\u003csup\u003e14\u003c/sup\u003e (\u003cstrong\u003eExtended Data Fig. 2\u003c/strong\u003e): acetoacetate (VirC), (\u003cem\u003eRS\u003c/em\u003e)-3-hydroxy-3-methylglutarate (VirD) and 3-methylcrotonate (VirE). The trends in relative affinities for acetoacetyl-ACP\u003csub\u003e5a\u003c/sub\u003e/\u003csub\u003e5b\u003c/sub\u003e and methylcrotonyl-ACP\u003csub\u003e5a\u003c/sub\u003e/\u003csub\u003e5b\u003c/sub\u003e were in line with those for the \u003cem\u003eholo\u003c/em\u003e proteins (\u003cstrong\u003eExtended Data Table 1, Supplementary Figs. 1\u003c/strong\u003e\u0026minus;\u003cstrong\u003e3\u003c/strong\u003e), consistent with the strong preference of the cassette for ACP\u003csub\u003e5b\u003c/sub\u003e, while the presence of substrate analogues did not increase but moderately diminished affinity. The latter result, which implies substrate tolerance, is encouraging for the prospect of generating analogues. Binding by VirD to HMG-ACP\u003csub\u003e5a\u003c/sub\u003e was likewise weaker than to HMG-ACP\u003csub\u003e5b\u003c/sub\u003e, although the difference was less marked than for the other analogues (51\u0026nbsp;\u0026plusmn;\u0026nbsp;25\u0026nbsp;mM vs. 39\u0026nbsp;\u0026plusmn;\u0026nbsp;5\u0026nbsp;mM) (\u003cstrong\u003eExtended Data Table 1, Supplementary Fig. 2\u003c/strong\u003e). We excluded an effect on binding to HMG-ACP\u003csub\u003e5b\u003c/sub\u003e of catalysis, by testing a catalytically-inactive version of VirD (E128Q\u003csup\u003e15\u003c/sup\u003e)\u0026nbsp;(\u003cstrong\u003eExtended Data Fig. 1\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. 2\u003c/strong\u003e), which yielded essentially the same K\u003csub\u003ed\u003c/sub\u003e (33\u0026nbsp;\u0026plusmn;\u0026nbsp;3\u0026nbsp;mM)\u0026nbsp;as for the wild type protein.\u0026nbsp;Overall, the bulk of the fluorescence quenching data are consistent with preferential binding by the three\u0026nbsp;b-methylation enzymes of ACP\u003csub\u003e5b\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterisation of the ACP\u003csub\u003e5b\u003c/sub\u003e/cassette interactions.\u0026nbsp;\u003c/strong\u003eTo gain insight into the determinants of interaction specificity, we aimed to solve the structures of complexes of ACP\u003csub\u003e5b\u003c/sub\u003e with VirC\u0026minus;VirE. However, we were unable to obtain crystals with wild type recombinant VirC and VirE (\u003cstrong\u003eExtended Data Fig. 1\u003c/strong\u003e, \u003cstrong\u003eSupplementary Tables 1\u003c/strong\u003e and \u003cstrong\u003e2\u003c/strong\u003e), nor with VirC quadruply mutated to promote crystallisation (C114A/Q334A/R335A/R338A) (\u003cstrong\u003eExtended Data Fig. 1\u003c/strong\u003e), as previously described for its homologue CurD from the curacin pathway\u003csup\u003e13\u003c/sup\u003e. Nonetheless, comparison of small-angle X-ray scattering (SAXS) data obtained on wild type VirC complexed with \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e, with that calculated\u003csup\u003e16\u003c/sup\u003e from the crystal structure of the acetyl-ACP\u003csub\u003eD\u003c/sub\u003e\u0026minus;CurD\u0026nbsp;complex (PDB: 5KP6)\u003csup\u003e13\u003c/sup\u003e, revealed a remarkable fit between the experimental and theoretical scattering curves (c\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 1.524)\u0026nbsp;(\u003cstrong\u003eExtended Data Fig. 3\u003c/strong\u003e, \u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e). This result shows that the overall structures are similar, implying that the ACP/partner recognition elements are likely to be shared between the two complexes. In the acetyl-ACP\u003csub\u003eD\u003c/sub\u003e\u0026minus;CurD\u0026nbsp;case\u003csup\u003e13\u003c/sup\u003e, the interface involves\u0026nbsp;the entirety of helix\u0026nbsp;a2, the loop\u0026nbsp;a2-a3 and helix\u0026nbsp;a3, as well as a key orientational interaction between the Ppant phosphate and CurD Arg33\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNext, we successfully solved the structure of VirD alone at 1.7\u0026nbsp;\u0026Aring; resolution (PDB ID: 8AHZ) (\u003cstrong\u003eFig. 2a\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e), as well as that of the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD complex by\u0026nbsp;Se-SAD at 2.1\u0026nbsp;\u0026Aring; (PDB ID:\u0026nbsp;8AHQ) (\u003cstrong\u003eFig. 2b\u003c/strong\u003e).\u0026nbsp;The final VirD model consists of a trimer in the asymmetric unit with r.m.s.d. between monomers of 0.2 \u0026Aring; (202 C\u003csub\u003ea\u003c/sub\u003e), whose solution relevance was confirmed by SAXS analysis (PRIMUS\u003csup\u003e17\u003c/sup\u003e) (\u003cstrong\u003eExtended Data Fig. 3\u003c/strong\u003e, \u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e). VirD belongs to the crotonase superfamily whose members exhibit a characteristic fold formed from repeated \u0026beta;\u0026beta;\u0026alpha; units\u003csup\u003e18\u003c/sup\u003e (\u003cstrong\u003eFig. 2\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD complex\u0026nbsp;(PDB ID: 8AHQ) (\u003cstrong\u003eFig. 2b\u003c/strong\u003e), the asymmetric unit contains two monomers of VirD and two of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e. As determined by the H3 crystal symmetry, VirD forms characteristic homotrimeric disks\u003csup\u003e18\u003c/sup\u003e, two of which are stacked, with six ACPs distributed equatorially at the interface between the trimers.\u0026nbsp;In this arrangement, the smallest gap between S6871 of ACP\u003csub\u003e5b\u003c/sub\u003e bearing the Ppant (~20\u0026nbsp;\u0026Aring;)\u0026nbsp;and the catalytic E128 of a VirD monomer is ca. 13.9\u0026nbsp;\u0026Aring; (\u003cstrong\u003eFig. 2c\u003c/strong\u003e), with the other VirD active sites more than 33.8\u0026nbsp;\u0026Aring; distant.\u0026nbsp;VirD elements contributing to the interface include the\u0026nbsp;b-strand\u0026nbsp;b10 and the subsequent loop (b10-a4) of one monomer, and the\u0026nbsp;b-turn (b1\u0026minus;b2), the loop (a1\u0026minus;b4) and helix\u0026nbsp;a7 of a second monomer (\u003cstrong\u003eFig. 2bc\u003c/strong\u003e)\u003cstrong\u003e.\u003c/strong\u003e Notably, the interface also incorporates the well-folded helix\u0026nbsp;a10 of the first monomer, which is disordered in the structure of VirD alone (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). Concerning the ACP, the interaction involves the C-terminal portion of helix\u0026nbsp;a1, the adjacent loop (a1\u0026minus;a2) and the N-terminal regions of helices\u0026nbsp;a2 and\u0026nbsp;a3. Specific interface residues include T6850 (helix\u0026nbsp;a1), Y6852 (loop\u0026nbsp;a1\u0026minus;a2), D6870, I6872, V6875 and E6876 (helix\u0026nbsp;a2) and Y6895 (helix\u0026nbsp;a3) (\u003cstrong\u003eFig. 2c\u003c/strong\u003e, \u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe structure of the complex also identifies key interactions between VirD and the Ppant tethered to S6871 of ACP\u003csub\u003e5b\u003c/sub\u003e (the distal end of which is not visible in the electron density (\u003cstrong\u003eFig. 2c\u003c/strong\u003e)), consistent with its contribution to binding affinity as observed by fluorescence quenching (\u003cstrong\u003eExtended Data Table 1\u003c/strong\u003e). Specifically, R125 of VirD, whose side chain is oriented by a water molecule, forms a salt bridge with the Ppant phosphate. The same water molecule bridges R192 from an adjacent VirD monomer, which participates in a salt bridge with D6870 of ACP\u003csub\u003e5b\u003c/sub\u003e. The opposite end of the D6870 carboxylate sits in an oxyanion hole comprising the NH groups of I6872 and L6873 of ACP\u003csub\u003e5b\u003c/sub\u003e helix\u0026nbsp;a2. Overall, these interactions place the oxygen of S6871 within\u0026nbsp;13.9\u0026nbsp;\u0026Aring; of the buried catalytic E128 of VirD, and thus within reach of the Ppant arm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, we turned our attention to VirE, studying its interaction with \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e in solution by SAXS combined with modelling using AlphaFold\u003csup\u003e19\u003c/sup\u003e (\u003cstrong\u003eExtended Data Fig. 3\u003c/strong\u003e, \u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e). Globally, this analysis indicates that VirD and VirE exhibit essentially the same overall folds and trimeric quaternary structures, and thus accordingly, the observed \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirE complex closely resembles that of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural basis for ACP\u003csub\u003e5b\u003c/sub\u003e/VirD interaction specificity and ACP anti-selection\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIdentification of the amino acids in ACP\u003csub\u003e5a\u003c/sub\u003e corresponding to the ACP\u003csub\u003e5b\u003c/sub\u003e interface residues shows that with only one exception (V6749 [ACP\u003csub\u003e5a\u003c/sub\u003e] vs. T6849 [ACP\u003csub\u003e5b\u003c/sub\u003e]), they are identical (\u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e). Thus, this set of residues does not constitute the basis for specific recognition of ACP\u003csub\u003e5b\u003c/sub\u003e by VirD. We therefore reassessed the hypothesis\u003csup\u003e7\u003c/sup\u003e that ACP recognition might derive, at least in part, from the relative orientation of the\u0026nbsp;a-helices within the domain structures. For this, we solved the NMR structures of\u0026nbsp;\u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e (PDB ID: 8A7Z), \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e6\u0026nbsp;\u003c/sub\u003e(PDB ID: 8AIG) and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u0026nbsp;\u003c/sub\u003e(PDB ID: 8ALL) (\u003cstrong\u003eSupplementary Table 5\u003c/strong\u003e), complementing the previously solved \u003cem\u003eapo\u003c/em\u003e-ACP\u003csub\u003e5b\u0026nbsp;\u003c/sub\u003e(PDB ID: 4CA3) and \u003cem\u003eapo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e (PDB ID: 2MF4) structures\u003csup\u003e10\u003c/sup\u003e. Superimposition of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e (PDB ID: 8AHQ) from the\u0026nbsp;\u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD\u0026nbsp;crystal structure (\u003cstrong\u003eFig. 2b\u003c/strong\u003e) with \u003cem\u003eapo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e, \u003cem\u003eapo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e, \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e, \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e6\u003c/sub\u003e and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u0026nbsp;\u003c/sub\u003ereveals r.m.s.d.\u0026nbsp;of 0.732 \u0026Aring; (74 Ca),\u0026nbsp;1.076 \u0026Aring; (59 Ca),\u0026nbsp;1.095 \u0026Aring; (72 Ca), 1.781 \u0026Aring; (72 Ca), 2.734\u0026nbsp;\u0026Aring;\u0026nbsp;(72\u0026nbsp;Ca), respectively.\u0026nbsp;ACPs 5a, 5b, 6 and 7 thus exhibit the same overall organisation including the orientation of the four\u0026nbsp;a-helices (\u003cstrong\u003eFig. 3a\u003c/strong\u003e), an architecture conserved with the previously-characterised Mup ACPs on which the Trp flag model was based\u003csup\u003e7\u003c/sup\u003e (\u003cstrong\u003eFig. 3b\u003c/strong\u003e).\u0026nbsp;Therefore, while the Trp provides strong predictive value for ACP sites of\u0026nbsp;b-branching\u003csup\u003e7\u003c/sup\u003e, our results argue against an important role for this residue and the resulting orientation of helix\u0026nbsp;a3, as determinants of cassette interaction specificity with ACP\u003csub\u003eA\u003c/sub\u003es. Indeed, both ACP\u003csub\u003e5a\u003c/sub\u003e and ACP\u003csub\u003e5b\u003c/sub\u003e contain Phe at this position instead of Trp. This observation is in line with further sequence variability recently uncovered at this position in other \u003cem\u003etrans\u003c/em\u003e-AT PKS systems\u0026nbsp;(\u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e)\u003csup\u003e20\u0026ndash;23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe origin of the observed minor differences in r.m.s.d. lies in the positions of the main chains of the loop regions, particularly\u0026nbsp;a1\u0026minus;a2. This observation prompted us to consider the potential contribution of the\u0026nbsp;a1\u0026minus;a2 loop to recognition. Gratifyingly, close inspection of the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD complex structure (\u003cstrong\u003eFig. 2c\u003c/strong\u003e) identified ACP\u003csub\u003e5b\u003c/sub\u003e N6865 located in the\u0026nbsp;a1\u0026minus;a2 loop as a potential specificity determinant. The\u0026nbsp;d-oxygen and nitrogen atoms of\u0026nbsp;N6865\u0026nbsp;hydrogen bond to two water molecules which are members of a larger, four-molecule water network forming hydrogen bonds to the main chain atoms of ACP\u003csub\u003e5b\u003c/sub\u003e residues\u0026nbsp;N6865, L6869, D6870, L6873, and L6894.\u0026nbsp;The constraints imposed by this network on the L6869 carbonyl, coupled with those on the D6870 side chain resulting from interaction with R192 of VirD and the ACP helix\u0026nbsp;a2 oxyanion hole, position the D6870 carboxylate at a distance of 3.9 \u0026Aring; from the phosphate of the Ppant arm.\u0026nbsp;The resulting position adopted by the Ppant to minimise electrostatic repulsion with D6870 apparently favours its efficient interaction with VirD. Notably, in ACP\u003csub\u003e5a\u003c/sub\u003e, polar N6865 is substituted by hydrophobic L6764\u0026nbsp;(\u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e),\u0026nbsp;a residue which cannot participate in the hydrogen bond network.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNonetheless, analysis of the ACP\u003csub\u003e6\u003c/sub\u003e and ACP\u003csub\u003e7\u003c/sub\u003e sequences reveals that the situation is more complicated than is evident from a single complex structure, as the equivalent sequence position in ACP\u003csub\u003e6\u003c/sub\u003e that does not interact with VirD is a Glu, while that in ACP\u003csub\u003e7\u003c/sub\u003e which is recognised, is also a Leu (\u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e). Thus, if a comparable water-mediated hydrogen bonding network is necessary to establish the correct orientation of the Ppant for binding VirD, other residues in ACP\u003csub\u003e7\u003c/sub\u003e can apparently substitute for the Asn of ACP\u003csub\u003e5b\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eInspection of the ACP structures also revealed that they diverge in terms of the pattern of charged, hydrophilic and hydrophobic residues on the surfaces adjacent to the Ppant arm\u0026nbsp;(\u003cstrong\u003eFig. 4\u003c/strong\u003e)\u003csup\u003e24\u003c/sup\u003e. Notably, in the case of ACP\u003csub\u003e5b\u003c/sub\u003e (\u003cstrong\u003eFig. 4a\u003c/strong\u003e), the surface surrounding the negatively-charged phosphate group of the Ppant and the adjacent, conserved acidic residue D6870, is largely hydrophobic but punctuated by a protruding hydrophilic region composed of S6863, N6865 and T6866. This region is itself encircled by three acidic patches, two contributed by the\u0026nbsp;a1\u0026minus;a2 loop (E6854, D6857; D6859, E6861), and the third located at the N-terminus of helix\u0026nbsp;a3 (D6896). ACP\u003csub\u003e7\u003c/sub\u003e exhibits an overall similar charge distribution to ACP\u003csub\u003e5b\u0026nbsp;\u003c/sub\u003e(\u003cstrong\u003eFig. 4b\u003c/strong\u003e). In this case, the hydrophilic patch is replaced by closely co-localised residues R2004, L2007 and E2008, while the ACP\u003csub\u003e5b\u003c/sub\u003e acidic patch comprising D6859 and E6861 is maintained by ACP\u003csub\u003e7\u003c/sub\u003e\u0026shy; residues D2001 and D2003. The surface additionally comprises an acidic residue D1995 unique to this domain.\u003c/p\u003e\n\u003cp\u003eIn contrast, in ACP\u003csub\u003e5a\u0026nbsp;\u003c/sub\u003e(\u003cstrong\u003eFig. 4c\u003c/strong\u003e), the hydrophilic protrusion is less extensive, and flanked by a hydrophobic region comprising F6763 and L6764, while one of the equivalent\u0026nbsp;a1\u0026minus;a2 loop patches contains the positively-charged R6756. In addition, residue A6862 in ACP\u003csub\u003e5b\u003c/sub\u003e is replaced by E6761 in ACP\u003csub\u003e5a\u003c/sub\u003e, contributing an additional negative charge to the surface (\u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e).\u0026nbsp;Consequently, when the residue at this position is small and hydrophobic it can participate in the ACP core, but when charged, the side chain points towards the solvent. ACP\u003csub\u003e6\u003c/sub\u003e differs even more dramatically from ACP\u003csub\u003e5b\u0026nbsp;\u003c/sub\u003e(\u003cstrong\u003eFig. 4d\u003c/strong\u003e). Specifically, the hydrophilic cluster is replaced by the acidic residue E1218 which is sandwiched between two cationic amino acids, R1206 and R1250, and uniquely among the four ACPs, the domain contains an additional positively-charged residue R1228 near the Ppant phosphate. Thus, both ACPs 5a and 6 exhibit positive net charge in regions which are negatively-charged in ACPs 5b and 7,\u0026nbsp;electrostatic features which we propose disfavour productive complex formation with the\u0026nbsp;b-cassette enzymes.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSite-directed mutagenesis supports the specificity model.\u0026nbsp;\u003c/strong\u003eTaken together, the obtained data suggested a model in which ACP recognition by VirD (and possibly all cassette members) depends principally on the subtle electrostatic landscape of the ACP surface which drives certain interactions, and potentially on the precise positioning of the Ppant arm within the resulting binary complexes, with only a minor role played by the attached substrates. To directly test this idea,\u0026nbsp;we exchanged\u0026nbsp;a1\u0026minus;a2 loop residues\u0026nbsp;E6761 and L6764 of ACP\u003csub\u003e5a\u003c/sub\u003e with their equivalents in ACP\u003csub\u003e5b\u003c/sub\u003e, A6862 (position contributing to the surface potential) and N6865 (surface hydrophilicity and/or Ppant orientation) (\u003cstrong\u003eExtended Data Figs. 1\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Supplementary Table 1\u003c/strong\u003e), and evaluated binding of the single and double mutants to VirD by tryptophan fluorescence quenching.\u0026nbsp;While VirD failed to bind \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e, it showed good affinity to both of the single \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u0026nbsp;\u003c/sub\u003emutants\u0026nbsp;(E6761A\u0026nbsp;[6.1\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u0026nbsp;mM] and\u0026nbsp;L6764N\u0026nbsp;[4.1\u0026nbsp;\u0026plusmn;\u0026nbsp;0.4\u0026nbsp;mM]), with K\u003csub\u003ed\u003c/sub\u003es comparable to those for binding \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e (\u003cstrong\u003eExtended Data Table 1\u003c/strong\u003e).\u0026nbsp;Thus, either single mutation results in VirD recognition.\u0026nbsp;Binding to the double mutant was also observed (7\u0026nbsp;\u0026plusmn;\u0026nbsp;1\u0026nbsp;mM),\u0026nbsp;albeit at slightly reduced affinity, perhaps due to minor perturbation of the ACP\u003csub\u003e5a\u003c/sub\u003e structure as judged by circular dichroism (\u003cstrong\u003eExtended Data Fig. 1\u003c/strong\u003e). It is also notable that VirD systematically exhibited higher affinity for the \u003cem\u003eholo\u003c/em\u003e form of the ACP\u003csub\u003e5a\u003c/sub\u003e mutants relative to the \u003cem\u003eapo\u003c/em\u003e forms (by 4\u0026minus;20-fold) (\u003cstrong\u003eExtended Data Table 1\u003c/strong\u003e), confirming the crucial role of the Ppant cofactor in the interaction.\u003c/p\u003e\n\u003cp\u003eWe also demonstrated that, in contrast to the lack of binding of wild type\u003cem\u003e\u0026nbsp;holo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e by VirC, both the E6761A\u0026nbsp;and L6764N \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e single mutants were recognised\u0026nbsp;(4.7\u0026nbsp;\u0026plusmn;\u0026nbsp;0.5\u0026nbsp;mM and 11\u0026nbsp;\u0026plusmn;\u0026nbsp;1\u0026nbsp;mM, respectively), while affinity to the double mutant was on par with that of L6764N\u0026nbsp;(10\u0026nbsp;\u0026plusmn;\u0026nbsp;1\u0026nbsp;mM) (\u003cstrong\u003eExtended Data Table 1\u003c/strong\u003e). These data support the idea that the\u0026nbsp;a1\u0026minus;a2 loop region of ACP\u003csub\u003e5b\u0026shy;\u003c/sub\u003e is also critical for its preferential recognition by VirC. Although the ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirC complex evidently resembles that of ACP\u003csub\u003eD\u003c/sub\u003e\u0026minus;CurD, understanding the detailed role played by these residues in the interaction awaits higher resolution structural information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of a doubly\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e-methylated virginiamycin derivative.\u0026nbsp;\u003c/strong\u003eThe observed binding between ACP\u003csub\u003e7\u003c/sub\u003e and the VirC\u0026minus;VirE implied that the ACP\u003csub\u003e7\u003c/sub\u003e-tethered intermediate may be targeted by the\u0026nbsp;b-methylation cassette \u003cem\u003ein vivo\u003c/em\u003e. To evaluate this idea, we scrutinised extracts of a second virginiamycin-producing strain, \u003cem\u003eStreptomyces pristinaespiralis\u003c/em\u003e ATCC 25486 (Sna cluster, \u003cstrong\u003eFig. 1\u003c/strong\u003e). In contrast to \u003cem\u003eS. virginiae\u003c/em\u003e, the complete genome sequence of \u003cem\u003eS. pristinaespiraelis\u003c/em\u003e is available, which is a necessary prerequisite for using CRISPR-Cas9\u003csup\u003e25\u003c/sup\u003e to verify the genetic origin of any detected metabolites, while avoiding off-target effects. To demonstrate the relevance of our interaction studies to this second strain, we measured binding between recombinant (\u003cstrong\u003eExtended Data Figs. 1\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e2\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Supplementary Tables 1\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e) \u003cem\u003eapo\u003c/em\u003e- and \u003cem\u003eholo\u003c/em\u003e-Sna ACP\u003csub\u003e7\u003c/sub\u003e and VirD. The sequence of VirD shows 63% identity to its Sna homologue, SnaJ\u003csup\u003e26\u003c/sup\u003e. Reassuringly, the K\u003csub\u003ed\u003c/sub\u003e determined for the most relevant \u003cem\u003eholo\u003c/em\u003e form (13\u0026nbsp;\u0026plusmn;\u0026nbsp;1\u0026nbsp;mM) was essentially identical to that measured for \u003cem\u003eholo\u003c/em\u003e-Vir ACP\u003csub\u003e7\u003c/sub\u003e, while that for the \u003cem\u003eapo\u003c/em\u003e-form was 2-fold lower (166\u0026nbsp;\u0026plusmn;\u0026nbsp;25 (Sna) vs.\u0026nbsp;75\u0026nbsp;\u0026plusmn;\u0026nbsp;7 (Vir)\u0026nbsp;mM)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eExtended Data Table 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eNext, LC-HRMS analysis of \u003cem\u003eS. pristinaespiraelis\u003c/em\u003e extracts revealed a signal at \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u0026nbsp;\u003c/em\u003e= 526.2912 ([M+H\u003csup\u003e+\u003c/sup\u003e]) (rt = 13.56; \u003cstrong\u003eFig. 5\u003c/strong\u003e), in excellent agreement with the calculated for potential analogues of Vir M \u003cstrong\u003e1\u003c/strong\u003e, incorporating a second\u0026nbsp;b-methyl at C-16 (\u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003eFig. 1c\u003c/strong\u003e). Masses corresponding to alternative doubly\u0026nbsp;b-methylated metabolites were not detected (\u003cstrong\u003eExtended Data Fig. 5\u003c/strong\u003e). Importantly, \u003cstrong\u003e3\u003c/strong\u003e was no longer detectable in S. \u003cem\u003epristinaespiralis\u0026nbsp;\u003c/em\u003eextracts when a portion of the module 7/module 8 interface was deleted using CRISPR-Cas9 (\u003cstrong\u003eExtended Data Fig. 6\u003c/strong\u003e, \u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e), directly confirming \u003cstrong\u003e3\u003c/strong\u003e as a product of the Sna pathway. Using commercial Vir M as a reasonable calibration standard (\u003cstrong\u003eSupplementary Fig.\u003c/strong\u003e \u003cstrong\u003e4\u003c/strong\u003e), we estimated the titres of \u003cstrong\u003e3\u003c/strong\u003e at 150\u0026minus;200-fold reduced relative to \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e2\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eExtended Data Table 2\u003c/strong\u003e). Therefore, while Vir ACP\u003csub\u003e7\u003c/sub\u003e is recognised with good affinity by the\u0026nbsp;b-methylation cassette, this modification is reduced under native biosynthetic conditions (\u003cstrong\u003eFig. 1a\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs the low absolute yields of \u003cstrong\u003e3\u003c/strong\u003e (\u003cstrong\u003eExtended Data Table 2\u003c/strong\u003e) precluded purification, to further support the structural assignment, we carried out comparative MS\u003csup\u003e2\u003c/sup\u003e analysis of \u003cstrong\u003e1\u003c/strong\u003e\u0026minus;\u003cstrong\u003e3\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eExtended Data Fig. 7\u003c/strong\u003e, \u003cstrong\u003eExtended Data Table 2\u003c/strong\u003e), and fed S. \u003cem\u003epristinaespiralis\u0026nbsp;\u003c/em\u003ecultures with isotopically-labelled amino acids, both individually and in combination:\u0026nbsp;L-proline-2,5,5-D\u003csub\u003e3\u003c/sub\u003e,\u0026nbsp;L-serine-2,3,3-D\u003csub\u003e3\u003c/sub\u003e and\u0026nbsp;L-proline-2,5,5-D\u003csub\u003e3\u003c/sub\u003e +\u0026nbsp;L-serine-2,3,3-D\u003csub\u003e3\u003c/sub\u003e. These amino acids were selected to confirm the relatedness of \u003cstrong\u003e3\u003c/strong\u003e to \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e2\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eFig. 1a\u003c/strong\u003e), and simultaneously track the post-incorporation chemistry via loss of deuterium. Comparison of the feeding data obtained on \u003cstrong\u003e3\u003c/strong\u003e to those of \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e2\u003c/strong\u003e (\u003cstrong\u003eExtended Data Fig. 8\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Supplementary Fig. 6\u003c/strong\u003e) demonstrates that \u003cstrong\u003e3\u003c/strong\u003e incorporates both Ser and Pro residues. Furthermore, the pattern of incorporation into \u003cstrong\u003e3\u003c/strong\u003e is consistent with retention of two deuteriums from both Ser and Pro. More specifically, the observed Pro labelling provides evidence for incorporation of\u0026nbsp;L-proline-2,5,5-D3 followed by dehydrogenation, as in \u003cstrong\u003e1\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eFig. 1c\u003c/strong\u003e). To explain the divergent labelling from Ser, we propose that Ser is incorporated by module 8 as normal, but that the subsequent HC-catalysed heterocyclisation/dehydrogenation does not occur due to mismatched substrate specificity. Indeed, the obtained MS\u003csup\u003e2\u003c/sup\u003e data are consistent with a structural difference between \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e3\u003c/strong\u003e in this region (\u003cstrong\u003eExtended Data Fig. 7\u003c/strong\u003e, \u003cstrong\u003eExtended Data Table 2\u003c/strong\u003e). Proline is then added by module 10, the product is liberated from the assembly line by macrocyclisation, and the Pro undergoes the native dehydrogenation reaction. Finally, transformation of the Ser to dehydroalanine may be catalysed spontaneously by an adventitious cellular enzyme, explaining the loss of the C-2 proton but retention of the two labels at C-3. Indeed, minute quantities of compound potentially corresponding to the non-dehydrated metabolite (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e = 544.3017; rt = 10.49 min) are also observed (\u003cstrong\u003eExtended Data Fig. 5\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDiverse\u0026nbsp;b-modification reactions occur during biosynthesis by many \u003cem\u003etrans\u003c/em\u003e-AT PKSs, and certain \u003cem\u003ecis\u003c/em\u003e-AT systems\u003csup\u003e3,27,28\u003c/sup\u003e. How specificity is achieved is an intriguing question, as the\u0026nbsp;b-modification cassette enzymes must distinguish between a large number of ACP\u003csub\u003eA\u003c/sub\u003e domains bearing potential\u0026nbsp;b-keto substrates. A further layer of complexity is the typical presence in\u0026nbsp;b-branching modules of 2\u0026minus;3 acceptor ACPs (ACP\u003csub\u003eA\u003c/sub\u003es), implying that one or all of these domains could serve as the site for the reaction series\u003csup\u003e29\u003c/sup\u003e. Understanding how acyl-ACP substrates are chosen, or conversely counter-selected, is a prerequisite to introducing\u0026nbsp;b-modification reactions at specific alternative positions in polyketides by genetic engineering.\u003c/p\u003e\n\u003cp\u003eIn this work, we investigated the\u0026nbsp;b-methylation module 5 present in the virginiamycin (Vir) \u003cem\u003etrans\u003c/em\u003e-AT PKS-NRPS, which comprises a KS domain and tandem ACPs (ACP\u003csub\u003e5a\u003c/sub\u003e and ACP\u003csub\u003e5b\u003c/sub\u003e) (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). Both ACP domains contain a Trp to Phe substitution at a residue position previously proposed to be critical for flagging the \u003cem\u003etrans\u003c/em\u003e-AT PKS ACP\u003csub\u003eA\u003c/sub\u003es at which\u0026nbsp;b-modification should occur\u003csup\u003e7\u003c/sup\u003e (\u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e), raising the questions of how they are recognised by the Vir cassette enzymes. Furthermore, the higher accessibility of ACP\u003csub\u003e5b\u003c/sub\u003e as revealed by the module 5 SAXS structure\u003csup\u003e10\u003c/sup\u003e, suggested that it might be the preferred site of\u0026nbsp;b-modification in \u003cem\u003etrans\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eWe show here that all members of the\u0026nbsp;b-methylation cassette do indeed preferentially recognise ACP\u003csub\u003e5b\u003c/sub\u003e, even when excised from its modular context, and that\u0026nbsp;b-modification occurs within defined ACP\u003csub\u003e5b\u003c/sub\u003e/partner complexes (\u003cstrong\u003eFig. 2\u003c/strong\u003e, \u003cstrong\u003eExtended Data Fig. 3\u003c/strong\u003e). The gate-keeping function within the cassette is therefore not limited to the HMGS VirC. The crystal structure of the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD complex (\u003cstrong\u003eFig. 2bc\u003c/strong\u003e) in combination with comparative sequence analysis (\u003cstrong\u003eExtended Data Fig. 4\u003c/strong\u003e), further reveals that the key ACP\u003csub\u003e5b\u003c/sub\u003e interface residues are highly conserved with ACP\u003csub\u003e5a\u003c/sub\u003e. ACP\u003csub\u003e5b\u003c/sub\u003e selectivity instead derives in large measure from the electrostatic character of the surrounding amino acids which drive complex formation\u003csup\u003e30\u003c/sup\u003e (\u003cstrong\u003eFig. 4\u003c/strong\u003e). Hydrogen-bonding restraints imposed on the Ppant cofactor (\u003cstrong\u003eFig. 2c\u003c/strong\u003e) may additionally optimise ACP/cassette interactions, but the attached substrates appear to contribute only minimally to the binding affinity\u003csup\u003e13\u003c/sup\u003e (\u003cstrong\u003eExtended Data Table 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eOverall, this specificity for ACP\u003csub\u003e5b\u003c/sub\u003e likely ensures that two ACPs act principally in-series to support, respectively, chain extension and\u0026nbsp;b-modification, which contrasts with previous reports of in-parallel function for such tandem ACPs\u003csup\u003e27,29,31\u003c/sup\u003e. This mechanism would require that the\u0026nbsp;b-keto intermediate be transacylated between the two ACPs, a transfer that is compatible with the measured inter-ACP distance\u003csup\u003e10\u003c/sup\u003e. It would also necessitate that the \u003cem\u003eholo\u003c/em\u003e form of ACP\u003csub\u003e5b\u0026nbsp;\u003c/sub\u003ebe present, but there is precedent for this in \u003cem\u003etrans\u003c/em\u003e-AT PKS systems\u003csup\u003e32\u003c/sup\u003e. While we can only speculate as to possible explanations, \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e may not be an efficient substrate for malonylation by the \u003cem\u003etrans\u003c/em\u003e-acting AT (VirI/SnaM), and/or it may be poorly accessible to the AT due to preassembly of complexes between ACP\u003csub\u003e5b\u003c/sub\u003e and the\u0026nbsp;b-methylation cassette enzymes.\u003c/p\u003e\n\u003cp\u003eGiven the prevailing view in the literature that\u0026nbsp;b-modification occurs with high fidelity\u003csup\u003e8\u003c/sup\u003e, we were surprised to observe that ACP\u003csub\u003e7\u003c/sub\u003e is also efficiently recognised by the three cassette enzymes \u003cem\u003ein vitro\u003c/em\u003e, an interaction which translates \u003cem\u003ein vivo\u003c/em\u003e into a previously-unidentified Vir M analogue \u003cstrong\u003e3\u003c/strong\u003e bearing a second\u0026nbsp;b-methyl group. Notably, titres of \u003cstrong\u003e3\u003c/strong\u003e at ca. 1% of those of \u003cstrong\u003e1\u003c/strong\u003e, are on par with amounts of polyketides typically obtained by PKS genetic engineering\u003csup\u003e2\u003c/sup\u003e. While this result might be interpreted as indicating that the pathway is intrinsically diversity-oriented, the fact that \u003cstrong\u003e3\u003c/strong\u003e titres are lower than those of \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e2\u003c/strong\u003e (\u003cstrong\u003eExtended Data Table 2\u003c/strong\u003e), rather argues that \u003cstrong\u003e3\u003c/strong\u003e arises from intermittent failures to suppress ACP\u003csub\u003e7\u003c/sub\u003e/cassette interactions. This phenomenon likely exemplifies the evolutionary challenges of achieving catalytic fidelity with acyl-ACP substrates which must interact with multiple partners, given the limited number of secondary structure and surface features offered by the small (ca. 10 kDa) domains\u003csup\u003e30\u003c/sup\u003e. This problem may be further aggravated by the evident structural plasticity of VirD (\u003cstrong\u003eFig. 2ab\u003c/strong\u003e) and VirE which derives from helix\u0026nbsp;a10, which could allow them to adapt to alternative partners. Furthermore, Module 7, which comprises only KS and ACP domains, is notably the sole PKS module in the Vir/Sna systems not to incorporate \u003cem\u003ecis\u003c/em\u003e-acting modification domains which could kinetically outcompete\u0026nbsp;b-methylation (\u003cstrong\u003eFig. 1a\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this context, we hypothesise that in addition to preferential recognition of ACP\u003csub\u003e5b\u003c/sub\u003e, the atypical\u003csup\u003e3\u003c/sup\u003e domain composition of module 8 also plays a role in\u0026nbsp;b-methylation programming. Notably, this module incorporates two copies of precisely the domains \u0026ndash; heterocyclisation (HC) and peptidyl carrier protein (PCP) (\u003cstrong\u003eFig. 1\u003c/strong\u003e) \u0026ndash; required for extension of the module 7 intermediate followed by oxazoline formation, suggesting that these domains kinetically and/or sterically outcompete the cassette enzymes, albeit imperfectly.\u0026nbsp;It may be noteworthy that kinetic arguments are now also used to explain complex programming in iterative PKSs\u003csup\u003e33\u003c/sup\u003e and NRPS systems with \u003cem\u003etrans\u003c/em\u003e-acting components\u003csup\u003e34\u003c/sup\u003e.\u0026nbsp;Given that a high proportion of \u003cem\u003etrans\u003c/em\u003e-AT PKSs systems comprise \u003cem\u003etrans\u003c/em\u003e-acting enzymes including but not limited to\u0026nbsp;b-branching cassettes\u003csup\u003e3,8\u003c/sup\u003e, it is likely that the existence of multiple control mechanisms is not limited to the virginiamycin system. We thus propose deblocking these latent chemistries as an innovative strategy for further diversifying polyketide structures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eOnline content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAny methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at \u003ca data-fr-linked=\"true\" href=\"https://doi.org\"\u003ehttps://doi.org\u003c/a\u003e...\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge financial support from\u0026nbsp;the Agence Nationale de la Recherche (grant numbers ANR-11-JSV8-003-01, PKS-PPIs;\u0026nbsp;ANR-16-CE92-0006-01, PKS STRUCTURE; and,\u0026nbsp;ANR-20-CE93-0002-01,\u0026nbsp;PKSOx to K.J.W.), the\u0026nbsp;Universit\u0026eacute; de Lorraine and\u0026nbsp;the Centre National de la Recherche Scientifique (CNRS).\u0026nbsp;We also acknowledge J. Davison for help with the molecular biology, Omar A. Rifi for assistance with production production and modification, and W. Shepard and M. Savko (Soleil Synchrotron, Proxima2) as well as J. Perez and A. Thureau (Soleil Synchrotron, Swing) for help with data acquisition. The NMR data were recorded on the NMR spectrometer of the Plateforme de Biophysique et Biologie Structurale (B2S) (IBSLor, UMS2008, CNRS-UL-INSERM). Analytical chemistry was performed on the\u0026nbsp;Structural and Metabolomics Analyses Platform (PASM), SF4242, Universit\u0026eacute; de Lorraine, EFABA, Vand\u0026oelig;uvre‐l\u0026egrave;s‐Nancy, France.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.G., K.J.W. and S.C. designed the study and carried out comparative sequence analysis. S.C. and B.C. designed and performed the molecular biology experiments. S.C. expressed and purified recombinant proteins, and S.C. and B.C. generated modified versions. S.C. performed the biophysical analyses, carried out the X-ray crystallography and SAXS analysis with A.G., and engineered the pathway inactivation with help from C.J. B.C. solved and analysed the ACP NMR structures. C.P. carried out the HPLC-MS experiments, and along with R.J.C., helped K.J.W. with data analysis and interpretation. All authors discussed the results. K.J.W., A.G. and S.C. wrote the manuscript, with input from BC.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended data\u0026nbsp;\u003c/strong\u003eis available for this paper at\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehttps://doi.org/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e. The online version contains supplementary material available at\u0026nbsp;https://doi.org/\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u0026nbsp;\u003c/strong\u003eshould be addressed to B.C., K.J.W. or A.G.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permission\u003c/strong\u003e information is available online at\u0026nbsp;\u003ca href=\"http://npg.nature.com/reprintsandpermissions/\"\u003ehttp://npg.nature.com/reprints\u003c/a\u003e\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hertweck, C. The biosynthetic logic of polyketide diversity. \u003cem\u003eAngew. Chem. Int. Ed. 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Rep.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 1029\u0026ndash;1045 (2018).\u003c/p\u003e\n\u003cp\u003e32.\u0026nbsp; \u0026nbsp;Masschelein, J. \u003cem\u003eet al.\u003c/em\u003e A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis. \u003cem\u003eNat. Chem.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 906\u0026ndash;912 (2019).\u003c/p\u003e\n\u003cp\u003e33. \u0026nbsp; Cox, R. J. Curiouser and curiouser: progress in understanding the programming of iterative highly-reducing polyketide synthases. \u003cem\u003eNat. Prod. Rep.\u003c/em\u003e (2022) doi:10.1039/d2np00007e.\u003c/p\u003e\n\u003cp\u003e34. \u0026nbsp; Kaniusaite, M. \u003cem\u003eet al.\u003c/em\u003e A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics. \u003cem\u003eChem. Sci.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 9466\u0026ndash;9482 (2019).\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis.\u0026nbsp;\u003c/strong\u003e\u003cem\u003etrans\u003c/em\u003e-AT PKSs containing\u0026nbsp;b-methylation modules were identified using refs.\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e and\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e. For comparative analysis of ACP domains, all PKS subunit sequences (with the exception of VirFG\u003csup\u003e10\u003c/sup\u003e) were retrieved from the Protein data base (http://www.ncbi.nlm.nih.gov/protein), and domain boundaries were established relative to the solved structures of Vir ACPs 5a and 5b (PDB IDs: 2MF4, 4CA3)\u003csup\u003e10\u003c/sup\u003e. Sequence alignments shown in figures were generated using the NPS@ web server\u0026nbsp;(\u003ca href=\"https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html\"\u003ehttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html\u003c/a\u003e)\u003csup\u003e35\u003c/sup\u003e and the figures created with ESPript\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and DNA manipulation.\u0026nbsp;\u003c/strong\u003eBiochemicals and media were purchased from VWR (glycerol, NaPi, NaCl, MgSO\u003csub\u003e4\u003c/sub\u003e), BD (tryptone, yeast extract), Thermo Fischer Scientific (Tris, EDTA), Euromedex (isopropyl \u0026beta;-D-1-thiogalactopyranoside; IPTG), and Sigma-Aldrich (betaine, imidazole,\u0026nbsp;Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), starch), and Roquette (corn steep).\u0026nbsp;L-proline-2,5,5-D\u003csub\u003e3\u003c/sub\u003e and\u0026nbsp;L-serine-2,3,3-D\u003csub\u003e3\u003c/sub\u003e were purchased from CDN Isotopes. The enzymes for genetic manipulation were purchased from Thermo Fisher Scientific. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin\u0026reg;\u0026nbsp;Gel and PCR Clean‑up or NucleoSpin\u0026reg;\u0026nbsp;Plasmid DNA kits (Macherey Nagel). Standard PCR reactions were performed with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific); and reactions were carried out on a Mastercycler Pro (Eppendorf). DNA sequencing was carried out by Eurofins.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrains and media.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) strains (\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e) were obtained from Novagen and were cultured in LB medium\u0026nbsp;(yeast extract 10 g L\u003csup\u003e-1\u003c/sup\u003e, tryptone 5 g L\u003csup\u003e-1\u003c/sup\u003e, NaCl 10 g L\u003csup\u003e-1\u003c/sup\u003e, adjusted to pH 7.0 with NaOH)\u0026nbsp;or on LB agar plates (LB medium supplemented with 20 g L\u003csup\u003e-1\u003c/sup\u003e agar) at 37 \u0026deg;C. \u003cem\u003eStreptomyces pristinaespiralis\u003c/em\u003e ATCC 25486 (DMSZ, Germany) and the derived mutants were sporulated on RP agar plates (20 g L\u003csup\u003e-1\u003c/sup\u003e starch, 20 g L\u003csup\u003e-1\u003c/sup\u003e soybean flour, 0.5 g L\u003csup\u003e-1\u003c/sup\u003e valine, 0.5 g L\u003csup\u003e-1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4,\u0026nbsp;\u003c/sub\u003e1 g L\u003csup\u003e-1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e \u0026times;\u0026nbsp;7H\u003csub\u003e2\u003c/sub\u003eO, 2 g L\u003csup\u003e-1\u003c/sup\u003e NaCl, 3 g L\u003csup\u003e-1\u003c/sup\u003e CaCO\u003csub\u003e3\u003c/sub\u003e, 20 g L\u003csup\u003e-1\u003c/sup\u003e agar in tap water) for 7 days at 30 \u0026deg;C. \u0026nbsp;All strains were maintained in 20% (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) glycerol and stored at\u0026nbsp;-80 \u0026deg;C. \u003cem\u003eE. coli\u003c/em\u003e ET12567/pUZ8002 was used for conjugation and appropriate antibiotics were added to LB liquid and agar cultures at the following concentrations: ampicillin 100 mg L\u003csup\u003e-1\u003c/sup\u003e, kanamycin 50 mg L\u003csup\u003e-1\u003c/sup\u003e, apramycin 25 mg L\u003csup\u003e-1\u003c/sup\u003e, chloramphenicol 25 mg L\u003csup\u003e-1\u003c/sup\u003e and nalidixic acid 25 mg L\u003csup\u003e-1\u003c/sup\u003e. For metabolite production by \u003cem\u003eS. pristinaespiralis\u003c/em\u003e and its mutant (\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e), 20 \u0026micro;L of spores were used to inoculate 25 mL inoculum medium (10 g L\u003csup\u003e-1\u003c/sup\u003e corn steep powder, 15 g L\u003csup\u003e-1\u003c/sup\u003e saccharose, 10 g L\u003csup\u003e-1\u003c/sup\u003e (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 1 g L\u003csup\u003e-1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 3 g L\u003csup\u003e-1\u003c/sup\u003e NaCl, 0.2 g L\u003csup\u003e-1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e \u0026times;\u0026nbsp;7H\u003csub\u003e2\u003c/sub\u003eO, 1.25 g L\u003csup\u003e-1\u003c/sup\u003e CaCO\u003csub\u003e3\u003c/sub\u003e in tap water, pH 6.9), followed by incubation at 30 \u0026deg;C and 180 rpm on rotary shaker for 72 h. Production medium (25 g L\u003csup\u003e-1\u003c/sup\u003e soybean flour, 7.5 g L\u003csup\u003e-1\u003c/sup\u003e starch, 22.5 g L\u003csup\u003e-1\u003c/sup\u003e glucose, 3.5 g L\u003csup\u003e-1\u003c/sup\u003e yeast extract, 0.5 g L\u003csup\u003e-1\u003c/sup\u003e ZnSO\u003csub\u003e4\u003c/sub\u003e \u0026times;\u0026nbsp;7H\u003csub\u003e2\u003c/sub\u003eO, 6 g L\u003csup\u003e-1\u003c/sup\u003e CaCO\u003csub\u003e3\u003c/sub\u003e in tap water, pH 6.0) was inoculated with 2% of precultures, and incubated at 30 \u0026deg;C, 180 rpm on a rotary shaker for 96 h. To evaluate its effect, certain cultures were supplemented with 2% XAD-16 resin (Sigma-Aldrich).\u0026nbsp;For feeding experiments, cultures were\u0026nbsp;supplemented individually with\u0026nbsp;L-proline-2,5,5-D\u003csub\u003e3\u003c/sub\u003e or\u0026nbsp;L-serine-2,3,3-D\u003csub\u003e3\u003c/sub\u003e, or a combination of\u0026nbsp;L-proline-2,5,5-D\u003csub\u003e3\u003c/sub\u003e and\u0026nbsp;L-serine-2,3,3-D\u003csub\u003e3\u003c/sub\u003e, at 4, 24 and 48 h after incubation, in equal portions, to a final concentration of 3 mM.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene cloning and site-directed mutagenesis.\u0026nbsp;\u003c/strong\u003eAll protein-encoding constructs were amplified directly from \u003cem\u003eStreptomyces virginiae\u003c/em\u003e genomic DNA using forward and reverse primers incorporating \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHind\u003c/em\u003eIII restriction sites, respectively (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e), and were ligated into the sites of vector pBG-102 for VirE and VirD and its mutant (VirD E128Q) or pLM-302 for VirC. Vector pBG-102 codes for a His\u003csub\u003e6\u003c/sub\u003e-SUMO tag and pLM-302 codes for a His\u003csub\u003e6\u003c/sub\u003e-maltose binding protein (MBP) tag (Centre for Structural Biology, Vanderbilt University).\u0026nbsp;Following cleavage of the tags, the proteins incorporated a non-native N-terminal Gly-Pro-Gly-Ser sequence. The sequences of all constructs were verified by DNA sequencing prior to protein expression studies.\u0026nbsp;Site-directed mutations were introduced into ACP\u003csub\u003e5a\u003c/sub\u003e and VirD\u0026nbsp;by PCR using mutagenic oligonucleotides\u0026nbsp;(\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e)\u0026nbsp;and Phusion High-Fidelity polymerase, followed by digestion of the parental DNA by 1 \u0026mu;L of \u003cem\u003eDpn\u003c/em\u003eI Fast digest (Thermo Fischer Scientific). The presence of the correct mutations was confirmed by sequencing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of recombinant proteins ACP domains, VirC, VirC quadruple mutant (C114A/Q334A/R335A/R338A), VirD, VirD E128Q and VirE.\u0026nbsp;\u003c/strong\u003eAll constructs were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells and grown at 37 \u0026deg;C in LB medium supplemented with 50\u0026nbsp;mg mL\u003csup\u003e-1\u003c/sup\u003e kanamycin to an A\u003csub\u003e600\u003c/sub\u003e of 0.8, and then IPTG added to a final concentration of 0.5 mM. Following incubation at 20 \u0026deg;C for 18 h, the cells were harvested by centrifugation at 3000\u003cem\u003eg\u003c/em\u003e for 30 min at 4 \u0026deg;C, and cell pellets stored immediately at\u0026nbsp;─80 \u0026deg;C. Vir ACP\u003csub\u003e5a\u003c/sub\u003e and ACP\u003csub\u003e5b\u003c/sub\u003e purification was performed as described previously\u003csup\u003e10\u003c/sup\u003e, and all APC\u003csub\u003e5a\u0026nbsp;\u003c/sub\u003e mutants, ACP\u003csub\u003e5a\u003c/sub\u003e─ACP\u003csub\u003e5b\u0026nbsp;\u003c/sub\u003edidomain, ACP\u003csub\u003e6\u003c/sub\u003e and ACP\u003csub\u003e7\u003c/sub\u003e purified using the same method.\u0026nbsp;In the case of all proteins of the\u0026nbsp;b-methylation cassette, the cell pellets were suspended in His-buffer (50 mM NaPi pH 7.5, 250 mM NaCl, 10 % glycerol for VirC and the VirC quadruple mutant, or 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 10 % glycerol (VirD, VirD E128Q and VirE)) containing 8 U mL\u003csup\u003e-1\u003c/sup\u003e of Benzonase (Merck) and 5 mM MgSO\u003csub\u003e4\u003c/sub\u003e. Cells were lysed by sonication and clarified by centrifugation (35,000 \u003cem\u003eg\u003c/em\u003e for 40 min).\u0026nbsp;Cell extracts were loaded onto a 5 ml HisTrap column (Cytiva) and washed with resuspension buffer supplemented with 20 mM imidazole. The supernatant was loaded onto a HisTrap 5 mL column equilibrated with His-buffer using an Akta Pure system (Cytiva). The proteins were eluted using a linear gradient of 0\u0026ndash;50% His-elution buffer (50 mM NaPi pH 7.5, 250 mM NaCl, 300 mM imidazole for VirC and the VirC quadruple mutant or 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 300 mM imidazole (VirD, VirD E128Q and VirE)) over ten column volumes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll His\u003csub\u003e6\u003c/sub\u003e-tagged constructs were then incubated with His-tagged human rhinovirus 3C protease (1 \u0026micro;M) for 12─16 h at 4 \u0026deg;C to cleave the affinity/solubility tags. The constructs were then separated from the remaining His-tagged proteins by loading onto a HisTrap 5 mL column, followed by elution in resuspension buffer containing 20 mM imidazole. VirD, VirD E128Q and VirE\u0026nbsp;were subsequently injected onto a Q-sepharose column (trimethylammonium on 6% agarose) equilibrated in buffer (20 mM Tris-HCl pH 8.5, 20 mM NaCl, 10 % glycerol). All proteins were then eluted using an NaCl gradient (100 mM─1 M) at 5 mL min\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Eluted fractions found to contain protein of the correct molecular weight as judged by SDS-PAGE analysis were pooled, concentrated using an Amicon Ultracel-10 (Merck Millipore) by centrifugation at 4000\u003cem\u003eg\u003c/em\u003e, and loaded onto a Superdex 200 16/60 (Cytiva) equilibrated with 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol (VirD, VirD E128Q and VirE) or a Superdex 75 16/60 column (Cytiva) (VirC and the VirC quadruple mutant).\u0026nbsp;Following a concentration step, the purity of the recombinant proteins was determined by SDS-PAGE\u0026nbsp;(\u003cstrong\u003eExtended Data Fig. 1\u003c/strong\u003e), and their concentrations were determined by NanoDrop (or Qubit for ACP\u003csub\u003e6\u003c/sub\u003e) (Thermo Scientific), with extinction coefficients calculated using the ExPASy ProtParam tool\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of labelled protein samples for structural biology.\u0026nbsp;\u003c/strong\u003eSeleniated\u003cs\u003e\u0026nbsp;\u003c/s\u003ewild type VirD was produced in M9 minimal medium (50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO4, 22 mM KH\u003csub\u003e2\u003c/sub\u003ePO4, 10 mM NaCl, 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl, adjusted to pH 7.2 with NaOH) for SAD/MAD phasing. Autoclaved M9 medium was supplemented with 50 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e of thiamine and riboflavin, 4 g L\u003csup\u003e\u0026minus;1\u0026nbsp;\u003c/sup\u003eglucose, 100 \u0026mu;M CaCl\u003csub\u003e2\u003c/sub\u003e, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 40 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e selenomethionine, and 40 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e of the 19 amino acids, based on the methionine biosynthesis inhibition method\u003csup\u003e38\u003c/sup\u003e. \u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e15\u003c/sup\u003eN-enriched Vir ACP\u003csub\u003e5a\u003c/sub\u003e, ACP\u003csub\u003e6\u003c/sub\u003e and ACP\u003csub\u003e7\u003c/sub\u003e were produced in M9 medium supplemented with \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl (0.5 g L\u003csup\u003e-1\u003c/sup\u003e) and \u003csup\u003e13\u003c/sup\u003eC-glucose (2.0 g L\u003csup\u003e-1\u003c/sup\u003e), as the only sources of nitrogen and carbon.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe labelled proteins were purified to homogeneity as described above.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSvp-catalysed modification of ACP domains and verification by HPLC-MS.\u0026nbsp;\u003c/strong\u003eFollowing size exclusion chromatography, \u003cem\u003eapo\u003c/em\u003e-ACPs (1 mM) were incubated in buffer (20 mM Tris-HCl pH 8.5) with 5 mM (acyl-)CoASH, 40\u0026nbsp;mM PPTase Svp\u003csup\u003e14\u003c/sup\u003e, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 50 mM TCEP for 22 h at 20 \u0026deg;C. The ACPs were then purified using a Superdex 75 16/60 column (Cytiva) equilibrated in 20 mM Tris-HCl pH 8.5, 250 mM NaCl, 50 mM TCEP. Quantitative modification was verified for all of the ACPs by HPLC-MS (\u003cstrong\u003eExtended Data Fig. 2\u003c/strong\u003e) using either a Thermo Scientific Orbitrap ID-X Tribrid Mass Spectrometer, or an LTQXL mass spectrometer, both equipped with an in-line photodiode array detector (PDA) and an atmospheric pressure ionization interface operating in electrospray mode (ESI). All samples were diluted with Milli-Q water to a concentration of 50 \u0026micro;M and injected onto an Alltima\u0026trade; C18 column (2.1 \u0026times; 150 mm, 5 \u0026micro;m particle size). Analysis was carried out with Milli-Q water containing 0.1% TFA (A) and acetonitrile containing 0.1% TFA (B), using the elution profile: 0\u0026minus;15 min, linear gradient from 10\u0026minus;98% solvent B; 15\u0026minus;20 min, constant 98% solvent B; 20.1\u0026minus;26 min, constant 10% solvent B. In the case of the LTQXL, MS scans were performed in ESI\u003csup\u003e+\u003c/sup\u003e in the mass range \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u0026nbsp;\u003c/em\u003e= 100\u0026minus;2000, at 3 K resolution, with MS parameters as follows: spray voltage, 5 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 20, 5 and 5 arbitrary units min\u003csup\u003e-1\u003c/sup\u003e; capillary temperature, 350 \u0026deg;C; capillary voltage, 7 V; tube lens, split lens and front lens voltages 180 V, ─22 V and ─11.75 V, respectively. MS data acquisition was carried out using the Xcalibur v. 2.1 software (Thermo Scientific). For the Orbitrap, MS scans were performed in heated ESI positive ion mode (HESI\u003csup\u003e+\u003c/sup\u003e) in the mass range \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e = 150\u0026minus;2000, at 7.5 K or 60 K resolution (full width of the peak at its half maximum, fwhm, at \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u0026nbsp;\u003c/em\u003e= 200) with MS parameters as follows: spray voltage, 4 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 30, 5 and 5 arbitrary units min\u003csup\u003e-1\u003c/sup\u003e; vaporiser and ion transfer tube temperatures were both set at 300 \u0026deg;C; maximum injection time, 50 ms; AGC target: 100000; normalised AGC target: 25%; microscans, 10; RF-lens, 35%; data type, profile. Mass spectrometer calibration was performed using the Pierce FlexMix calibration solution (Thermo Scientific). MS data acquisition was carried out using the Xcalibur v. 4.3 software (Thermo Scientific). For data obtained at low resolution (3 or 7.5 K), only the major isotopic peak was detected, while analysis at high resolution (60K) afforded the full isotopic spectrum (\u003cstrong\u003eExtended Data Fig. 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTryptophan fluorescence quenching.\u0026nbsp;\u003c/strong\u003eAll tryptophan fluorescence spectroscopy experiments were performed on a SAFAS Fluorescence Xenius Spectrophotometer (SAFAS, France) in a 2 mL quartz cuvette. The excitation wavelength was fixed at 295 nm and emission spectra were collected between 300\u0026minus;400 nm with a slit width of 2 nm. The\u0026nbsp;temperature was maintained at 25 \u0026deg;C by an external thermostatic water circulator. To measure protein-ligand interactions, recombinant VirC, VirD, VirD E128A mutant and VirE at 5\u0026nbsp;mM were allowed to equilibrate in TE buffer (20 mM Tris-HCl pH 8.5, 2 mM EDTA)\u0026nbsp;for 10 min under constant stirring, before being titrated with ligand solutions. The proteins were analysed against increasing concentrations of ligand (0─150\u0026nbsp;mM), depending on the specific ligand used.\u0026nbsp;Data from two\u0026nbsp;independent experiments were analysed using nonlinear regression, with application of the one site-specific binding model (F = F\u003csub\u003emax\u003c/sub\u003e*X / (K\u003csub\u003ed\u003c/sub\u003e + X), where X is the ligand concentration, F is the fluorescence intensity, F\u003csub\u003emax\u003c/sub\u003e is the maximum specific binding and K\u003csub\u003ed\u003c/sub\u003e is the equilibrium binding constant) using SciDAVis v2.3.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircular dichroism measurements.\u0026nbsp;\u003c/strong\u003eCircular dichroism measurements were performed on a Chirascan CD (Applied Photophysics) in 100 mM NaPi, 150 mM NaF pH 8.0. Data were collected at 0.5 nm intervals in the wavelength range of 180─260 nm at 20 \u0026deg;C, using a temperature-controlled chamber.\u0026nbsp;30\u0026nbsp;mL of 100\u0026nbsp;mM\u0026nbsp;ACP\u003csub\u003e5a\u003c/sub\u003e, ACP5a\u0026nbsp;E6761A/L6764N and VirD were analysed in a\u0026nbsp;0.01 cm cuvette, while 100\u0026nbsp;mL of\u0026nbsp;100\u0026nbsp;mM\u0026nbsp;VirD E128Q was analysed in a 0.1 cm cuvette. Each spectrum represents the average of three scans, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone. Spectrum deconvolution was carried out using the CDNN2.1 software\u003csup\u003e39\u003c/sup\u003e (\u003cstrong\u003eExtended Data Fig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall-angle X-ray scattering (SAXS) data collection.\u0026nbsp;\u003c/strong\u003eSAXS data were\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eacquired on the SWING beamline at the Synchrotron SOLEIL\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(France). The frames were recorded using an Eiger 4M detector at an energy of 12 keV. The distance between the sample\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand the detector was set to 2000 mm for VirD, VirE, \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirC and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirE complexes, leading to scattering vectors\u0026nbsp;\u003cem\u003eq\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eranging from 0.0005\u0026minus;0.5 \u0026Aring;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e. The scattering vector is defined as 4p/l\u0026nbsp;sinq, where 2q\u0026nbsp;is the scattering angle. The protein samples were\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003einjected using the online automatic sample changer into a pre-equilibrated\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHPLC-coupled size-exclusion chromatography column\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Bio-SEC 100\u0026nbsp;\u0026Aring;, Agilent), at a temperature of 15\u0026nbsp;\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe eluted fractions were delivered using an online purification system developed on the SWING beamline\u003csup\u003e40\u003c/sup\u003e. After equilibrating the column in the protein buffer (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol), the buffer background was recorded (100 successive frames of 0.75 s). A 50\u0026nbsp;mL aliquot of the protein sample (at 5 mg mL\u003csup\u003e-1\u003c/sup\u003e) was then injected, and complete data sets were collected. The protein concentration downstream of the elution column was followed via the absorbance at 280 nm with an\u0026nbsp;\u003cem\u003ein situ\u003c/em\u003e spectrophotometer. In lieu of analysing several protein concentrations within a standard range (\u003cem\u003ee.g.\u003c/em\u003e, 0.1\u0026minus;10 mg mL\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e), the coupling of data collection to a gel filtration column allows analysis of multiple concentrations of protein within a single experiment, as many distinct positions within the elution peak are sampled during the course of the measurement (typically 50\u0026minus;100 frames are acquired)\u003csup\u003e40\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing on from this, the dedicated in-house application FOXTROT was used to perform data reduction to absolute units, frame averaging, and solvent subtraction. Each acquisition frame of the experiment yielded a scattering spectrum, which was then analysed by FOXTROT to produce an\u0026nbsp;\u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(radius of gyration) as well as an\u0026nbsp;\u003cem\u003eI\u003c/em\u003e(0) value (the \u003cem\u003eI\u003c/em\u003e(0) depends on the protein concentration at that position in the elution peak, as described by the Guinier law (approximation\u0026nbsp;\u003cem\u003eI\u003c/em\u003e(\u003cem\u003eq\u003c/em\u003e)\u0026nbsp;= \u003cem\u003eI\u003c/em\u003e(0) exp(\u0026minus;\u003cem\u003eq\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e/3) for\u0026nbsp;\u003cem\u003eqR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e \u0026lt; 1.3). Notably, observing a constant\u0026nbsp;\u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e for a significant proportion of the concentrations present in the gel\u0026nbsp;filtration peaks showed that the measurements were concentration-independent, and thus that they were effectively carried out under conditions of infinite dilution.\u003c/p\u003e\n\u003cp\u003eFinally, all the frames exhibiting identical\u0026nbsp;\u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e as a function of\u0026nbsp;\u003cem\u003eI\u003c/em\u003e(0) were corrected for buffer signal and averaged. This step ensured that the data reflected only the signal arising from the protein structure and not from intermolecular interactions. The distance distribution function\u0026nbsp;\u003cem\u003eP\u003c/em\u003e(\u003cem\u003er\u003c/em\u003e) and the maximum particle diameter\u0026nbsp;\u003cem\u003eD\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e were then calculated by Fourier inversion of the scattering intensity\u0026nbsp;\u003cem\u003eI\u003c/em\u003e(\u003cem\u003eq\u003c/em\u003e) using GNOM\u003csup\u003e41\u003c/sup\u003e. The SAXS data are presented in \u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular weights and oligomeric structures in solution from SAXS data.\u0026nbsp;\u003c/strong\u003eClassically,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003emolecular weights can be derived from SAXS data using the\u0026nbsp;\u003cem\u003eI\u003c/em\u003e(0)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand the known protein concentration. However, this method was not appropriate in our case, as the delay between exiting the gel filtration column and the SAXS data acquisition may have altered the concentrations. We therefore determined the molecular weights of the constructs using Bayesian Interference in PRIMUS\u003csup\u003e42\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSAXS data were recorded on wild type VirE, as well as VirC and VirE complexed with \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e.\u0026nbsp;A model of a trimer of VirE was generated using ColabFold: AlphaFold2\u003csup\u003e19\u003c/sup\u003e. On the basis of the structural homology between the VirE model and the solved VirD crystal structure (r.m.s.d. 3.38 calculated based on 200 C\u003csub\u003ea\u003c/sub\u003e), we generated a model of the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirE complex by superimposition on the crystal structure of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD using PyMOL\u003csup\u003e43\u003c/sup\u003e.\u0026nbsp;The quality of the 3D modelling was determined using CRYSOL\u003csup\u003e16\u003c/sup\u003e to compare the fit between the theoretical scattering curves from atomic coordinates with experimental scattering curves, and judged using the discrepancy\u0026nbsp;c\u003csup\u003e2\u003c/sup\u003e, defined according to Konarev and colleagues\u003csup\u003e17\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSAXS data obtained on wild type VirC complexed with \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e were directly compared with that calculated\u003csup\u003e16\u003c/sup\u003e from the crystal structure of the acetyl-ACP\u003csub\u003eD\u003c/sub\u003e\u0026minus;CurD complex (PDB: 5KP6)\u003csup\u003e13\u003c/sup\u003e using CRYSOL\u003csup\u003e16\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrystallisation and X-ray data collection.\u0026nbsp;\u003c/strong\u003eSe-VirD was purified and stored in buffer (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol)\u0026nbsp;at a final concentration of 5 mg mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e. \u003cem\u003eholo\u003c/em\u003e ACP\u003csub\u003e5b\u003c/sub\u003e was stored in buffer (20 mM Tris-HCl pH 8.5, 250 mM NaCl, 50 mM TCEP) at\u0026nbsp;a final concentration of 20 mg mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Prior to crystallization trials, sample homogeneity was checked by dynamic light scattering (DLS) using a Zetasizer NanoS (Malverne). Initial crystallisation hits were obtained using the Rigaku kit (Molecular Dimensions). The conditions consisted of 20% PEG 400, 20% PEG 800, 100 mM Tris-HCl, pH 7.5 for Se-VirD, while \u003cem\u003eholo\u0026nbsp;\u003c/em\u003eACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;Se-VirD crystallised in 100 mM chloride calcium, 30% PEG 1500, 10% 2-propanol, 100 mM imidazole-HCl, pH 6.5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCrystals grew in 10\u0026minus;15 days using the hanging drop method in Linbro\u0026reg; plates, with drops formed by mixing 2\u0026nbsp;mL of protein solution (ratio 1:4 for the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;Se-VirD\u0026nbsp;complex, 5 mg mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e Se-VirD) with 1\u0026nbsp;mL of crystallisation buffer. Crystals were then soaked in crystallisation buffer containing 30% ethylene glycol prior to freezing in liquid nitrogen. X-ray diffraction data on Se-VirD and the \u003cem\u003eholo\u003c/em\u003e ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;Se-VirD complex were collected at the SOLEIL synchrotron on\u0026nbsp;the Proxima2 beamline. The crystals belong to the P4\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e2 and H3 space groups, respectively (\u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e). A complete MAD data set at four wavelengths was collected in order to solve the crystal structure of VirD. Data sets were indexed and integrated using XDS\u003csup\u003e44\u003c/sup\u003e and scaled by\u0026nbsp;using pointless and aimless (CCP4 package).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructure determination and refinement.\u003c/strong\u003e Initial\u0026nbsp;phases were ultimately generated via SAD using the peak wavelength (\u0026lambda; = 0.979260 \u0026Aring;). Three high confidence Se sites were identified and refined by using the NCS using Phenix.autosol\u003csup\u003e45,46\u003c/sup\u003e. The figure of merit (FOM) from Phenix AutoSol is 0.32. Density modification and NCS were then used to improve the quality of the phases (FOM: 0.68 with a bias ratio of 1.36). The good quality of the electron density map allowed for building approximatively 80% of the backbone at 2.02 \u0026Aring; using Phenix.autobuild\u003csup\u003e47\u003c/sup\u003e. The final model of WT VirD was built using ARP/wARP\u003csup\u003e48\u003c/sup\u003e, followed by iterative cycles of manual rebuilding and refinement at 1.7\u0026nbsp;\u0026Aring;\u0026nbsp;using COOT\u003csup\u003e49\u003c/sup\u003e and REFMAC5\u003csup\u003e50\u003c/sup\u003e.\u0026nbsp;The structure of the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD\u0026nbsp;complex was solved\u0026nbsp;by molecular replacement using a monomer of VirD as search model with the program MOLREP in CCP4\u003csup\u003e51,52\u003c/sup\u003e. The contrasted solution with final CC of 0.7252 and Tf/sig of 27.17, consists of 2 monomers of VirD in the asymmetric unit. The initial model was then refined by rigid body refinement at 3 \u0026Aring; followed by a restraint refinement at 2.1 \u0026Aring; resolution using REFMAC 5 CCP4\u003csup\u003e50\u003c/sup\u003e. The excellent quality of the electron density maps allowed us to locate two extra electron density in the F\u003csub\u003eo\u003c/sub\u003eF\u003csub\u003ec\u003c/sub\u003e map corresponding to two ACP\u003csub\u003e5b\u003c/sub\u003e molecules in the asymmetric unit. The ACPs were then constructed manually in the electron density maps. Structure geometry was validated using the program MolProbity\u003csup\u003e53\u003c/sup\u003e. The structures of VirD and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD contain 99.26% and 97.91% of the residues in the allowed region of the Ramachandran plot respectively and contain no outliers (\u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e). Figures were prepared using the program PyMOL\u003csup\u003e43\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein NMR data acquisition.\u0026nbsp;\u003c/strong\u003eAll ACPs proteins samples were buffer exchanged via gel filtration into 100 mM sodium phosphate (pH 6.0), 1 mM EDTA and 1 mM TCEP, concentrated to 1 mM, and then 350 \u0026mu;L of the samples (including 10% D\u003csub\u003e2\u003c/sub\u003eO) were loaded into 4 mm NMR tubes. All NMR data were recorded at 25 \u0026deg;C on a Bruker DRX600 spectrometer equipped with a cryogenic probe (Unit\u0026eacute; Mixte de Service (UMS) 2008 Ing\u0026eacute;nierie-Biologie-Sant\u0026eacute; en Lorraine\u0026nbsp;(IBSLor)). Backbone and sequential resonance assignments were obtained by the combined use of 2D \u003csup\u003e15\u003c/sup\u003eN\u0026minus;\u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC\u0026minus;\u003csup\u003e1\u003c/sup\u003eH HSQC spectra and 3D HNCA, HNCACB, CBCA(CO)NH, HNHA, HBHA(CO)NH, HN(CA)CO, and HNCO experiments. Assignments of aliphatic side chain resonances were based on 2D aromatic \u003csup\u003e13\u003c/sup\u003eC\u0026minus;\u003csup\u003e1\u003c/sup\u003eH HSQC, (HB)CB(CGCDCE)HE, (HB)CB(CGCD)HD and 3D (H)CC(CO)NH, H(CC)(CO)NH, CCH\u0026minus;TOCSY, and HCCH-TOCSY experiments (reviewed in\u0026nbsp;\u003csup\u003e54\u003c/sup\u003e). To collect NOE-based distance restraints for the structure calculations, 3D \u003csup\u003e15\u003c/sup\u003eN NOESY-HSQC and \u003csup\u003e13\u003c/sup\u003eC NOESY-HSQC were recorded on uniformly \u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e15\u003c/sup\u003eN enriched samples using a mixing time of 120 ms. NMR data were processed using Topspin 3.2 (Bruker) and were analysed using NMRFAM-SPARKY\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein NMR structure calculations.\u0026nbsp;\u003c/strong\u003eInitial structures were generated using CYANA 3.98 software\u003csup\u003e56\u003c/sup\u003e. Starting from a set of manually-assigned NOEs, the standard CYANA protocol of seven iterative cycles of calculations was performed with NOE assignment by the embedded CANDID routine combined with torsion angle dynamics structure calculation\u003csup\u003e57\u003c/sup\u003e. In each cycle, 100 structures starting from random torsion angle values were calculated with 15,000 steps of torsion angle dynamics-driven simulated annealing. A total of 1822, 1208 and 1763 NOE-based distances, 110, 92 and 94 backbone angle restraints were used for structure calculation of the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e, \u003cem\u003eholo-\u003c/em\u003eACP\u003csub\u003e6\u003c/sub\u003e and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e domains, respectively (\u003cstrong\u003eSupplementary Table 5\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe angle restraints were obtained from \u003csup\u003e13\u003c/sup\u003eC\u0026alpha;, \u003csup\u003e13\u003c/sup\u003eC\u0026beta;, \u003csup\u003e13\u003c/sup\u003eC\u0026prime;, \u003csup\u003e15\u003c/sup\u003eN, \u003csup\u003e1\u003c/sup\u003eHN, and \u003csup\u003e1\u003c/sup\u003eH\u0026alpha; chemical shifts using TALOS-N\u003csup\u003e58\u003c/sup\u003e with an assigned minimum range of \u0026plusmn;20\u0026deg;. 4\u0026cent;-phosphopantetheine-serine was created as a serine modified residue within the CYANA library using 4\u0026cent;-phosphopantetheine coordinates from the solution structure of \u003cem\u003eholo\u003c/em\u003e-ACP PfACP from \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (PDB ID: 2FQ0)\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe second stage consisted of the refinement of the 50 lowest CYANA target function conformers by restrained molecular dynamic (rMD) simulations in Amber 14\u003csup\u003e60,61\u003c/sup\u003e,\u0026nbsp;following published protocols\u003csup\u003e62\u003c/sup\u003e. Phosphopantetheinyl serine library and force field parameters\u003csup\u003e63\u003c/sup\u003e were used for AMBER minimisation.\u0026nbsp;The final representative ensembles correspond to the 20 conformers from each calculation with the lowest restraint energy terms. The structures of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e, \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e6\u003c/sub\u003e and \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e contain 98.6%, 94.4% and 97.1% in the most favoured region and 1.4 %, 5.6% and 2.9% of the residues (non-glycine and non-proline) in the additional allowed region of the Ramachandran plot, respectively. \u0026nbsp;PROCHECK statistics were calculated using PROCHECK-NMR\u003csup\u003e64\u003c/sup\u003e. The proportion of residues in the most favoured/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot for the ACPs are as follows: \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e (97.1/2.9/0/0); \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e\u0026shy;6\u003c/sub\u003e (94.3/5.7/0/0); \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u003c/sub\u003e (92.4/7.1/0.1/0.4).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of \u003cem\u003eS. pristinaespiralis\u003c/em\u003e pathway inactivation mutant.\u0026nbsp;\u003c/strong\u003eFor construction of the pathway mutant, the pCRISPomyces-2 plasmid\u003csup\u003e25\u003c/sup\u003e was used for CRISPR-Cas9-based genome editing. Spacer sequences (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e) were chosen using the online CRISPy-web software\u003csup\u003e65\u003c/sup\u003e, and were generated by annealing two 24 nt oligonucleotides. Next, 1 kb homologous arms HAL and HAR were amplified by PCR, the pCRISPomyces-2 plasmid was linearised with the restriction enzyme \u003cem\u003eXba\u003c/em\u003eI (Thermo Fisher Scientific), and then assembly of the editing templates and the pCRISPomyces-2 plasmid was performed using the In-Fusion HD Cloning kit (Ozyme, France). Correct plasmid assembly was confirmed by diagnostic digestion and sequencing (\u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e). Recombinant plasmids were introduced into \u003cem\u003eE. coli\u003c/em\u003e 12567 (pUZ8002) by electroporation. Conjugation of plasmids into \u003cem\u003eStreptomyces\u003c/em\u003e spores was performed using the protocol described elsewhere\u003csup\u003e66\u003c/sup\u003e. Following conjugation, clearance of the plasmid was accomplished by repeated high-temperature cultivation (37 \u0026deg;C) for 2\u0026ndash;3 days, followed by replica plating on selective and nonselective plates to confirm restoration of apramycin sensitivity. Apramycin-sensitive colonies were then picked into liquid ISP2 medium (4 g L\u003csup\u003e-1\u003c/sup\u003e yeast extract, 4 g L\u003csup\u003e-1\u003c/sup\u003e dextrose,\u0026nbsp;10 g L\u003csup\u003e-1\u003c/sup\u003e malt extract\u0026nbsp;adjusted to pH 7.3 with NaOH)\u0026nbsp;for genomic DNA isolation using the Wizard Genomic DNA Purification Kit (Promega). Genomic modifications were confirmed by PCR and sequencing of the modified regions\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis by HPLC-MS of the \u003cem\u003eS. pristinaespiralis\u003c/em\u003e pathway inactivation mutant.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eS. pristinaespiralis\u003c/em\u003e cultures were extracted\u0026nbsp;twice with ethyl acetate (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e).\u0026nbsp;When present, XAD-16 resin was harvested by sieving, and also extracted\u0026nbsp;twice with ethyl acetate (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e).\u0026nbsp;The solvent was removed by evaporation, the extracts resuspended in 1:1 ACN/water (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) and then the sample was passed through a 0.4 \u0026micro;m syringe filter. HPLC-MS analysis was performed in positive and/or negative electrospray mode (ESI+/\u0026minus;) on the Thermo Scientific Orbitrap ID-X Tribrid Mass Spectrometer using an Alltima\u0026trade; C18 column (2.1 \u0026times; 150mm, 5 \u0026micro;m particle size) at 25\u0026deg;C (flow rate, 0.2 mL min\u003csup\u003e-1\u003c/sup\u003e). Separation was carried out with Milli-Q water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B), using the following elution profile: 0\u0026minus;48 min, linear gradient 5\u0026minus;95% solvent B; 48\u0026minus;54 min, constant 95% solvent B; 54\u0026minus;60 min, constant 5% solvent B. Mass spectrometry operating parameters were as described previously. Metabolite yields\u0026nbsp;(\u003cstrong\u003eExtended Data Table 2\u003c/strong\u003e) were estimated by generating a calibration curve using commercially-available virginiamycin M \u003cstrong\u003e1\u003c/strong\u003e (Sigma-Aldrich), over the concentration range of\u0026nbsp;0.00128\u0026minus;20 mg L\u003csup\u003e-1\u003c/sup\u003e (10\u0026nbsp;mL of each sample was injected). This approach afforded a linear correlation between the quantity of metabolite\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand the respective integrated peak area in the extracted ion chromatogram (EIC) (the areas of the peaks corresponding to the parental ions [M+H]\u003csup\u003e+\u003c/sup\u003e were used systematically) (\u003cstrong\u003eSupplementary Fig. 4\u003c/strong\u003e). For analysis of metabolite yields in extracts, following conversion of peak areas to titres, the results were divided by 200 to correct for the enrichment of the sample during preparation, as the extracts from 20 mL of culture were resuspended in 100\u0026nbsp;mL of solvent prior to HPLC-MS analysis (as with the standard, 10\u0026nbsp;mL of each sample was injected).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCrystal structures of VirD and the \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5b\u003c/sub\u003e\u0026minus;VirD complex have been deposited in the Protein Data Bank with their respective diffraction data under accession codes 8AHZ and 8AHQ, respectively. Coordinates and chemical shifts for the NMR structures of \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e5a\u003c/sub\u003e, \u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e6\u0026nbsp;\u003c/sub\u003eand\u0026nbsp;\u003cem\u003eholo\u003c/em\u003e-ACP\u003csub\u003e7\u0026nbsp;\u003c/sub\u003ehas been deposited in the Biological Magnetic Resonance Bank with accession codes 8A7Z, 8AIG, and 8ALL, respectively.\u003csub\u003e\u0026nbsp;\u003c/sub\u003eSAXS and HPLC-MS data have been deposited\u0026nbsp;in the data repository DOREL (Donn\u0026eacute;es de la Recherche Lorraines) [\u003ca href=\"https://doi-org.insis.bib.cnrs.fr/10.12763/PEYXHP\"\u003ehttps://doi-org.insis.bib.cnrs.fr/10.12763/PEYXHP\u003c/a\u003e].\u0026nbsp;The remaining data supporting this study are included in the Supplementary Information. Source data are provided with this paper, and all biological materials are available from the authors upon request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e35.\u0026nbsp; \u0026nbsp;Thompson, J. D., Higgins, D. G. \u0026amp; Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 4673\u0026ndash;4680 (1994).\u003c/p\u003e\n\u003cp\u003e36.\u0026nbsp; \u0026nbsp;Gouet, P., Robert, X. \u0026amp; Courcelle, E. 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Microbiol.\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 792\u0026ndash;798 (2012).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2103032/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2103032/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"During biosynthesis by multi-modular trans-AT polyketide synthases (PKSs), polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase (HMGS) cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT PKS. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation, identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, imperfections in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.","manuscriptTitle":"Decrypting the programming of β-methylation in virginiamycin M biosynthesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-17 14:06:14","doi":"10.21203/rs.3.rs-2103032/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"213d4124-60b5-4794-ac60-9913d37b2185","owner":[],"postedDate":"October 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-03-11T08:08:54+00:00","versionOfRecord":{"articleIdentity":"rs-2103032","link":"https://doi.org/10.1038/s41467-023-36974-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2023-03-10 05:00:00","publishedOnDateReadable":"March 10th, 2023"},"versionCreatedAt":"2022-10-17 14:06:14","video":"","vorDoi":"10.1038/s41467-023-36974-3","vorDoiUrl":"https://doi.org/10.1038/s41467-023-36974-3","workflowStages":[]},"version":"v1","identity":"rs-2103032","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2103032","identity":"rs-2103032","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}