Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family

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Andersson , View ORCID Profile Maria Selmer doi: https://doi.org/10.1101/2020.08.16.253161 Xiaohu Guo 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden 5 Division of Biochemistry, Cancer Genomics Center, Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Annika Söderholm 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sandesh Kanchugal P 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sandesh Kanchugal P Geir Villy Isaksen 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden 2 Hylleraas Centre for Quantum Molecular Sciences, Department of Chemistry, UiT - The Arctic University of Norway , N9037, Tromsø, Norway Find this author on Google Scholar Find this author on PubMed Search for this author on this site Omar Warsi 3 Department of Medical Biochemistry and Microbiology, Uppsala University , BMC, Box 582, 751 23 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ulrich Eckhard 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden 6 Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona , CSIC, Barcelona Science Park, Baldiri Reixac, 15-21, 08028 Barcelona, Catalonia, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ulrich Eckhard Silvia Trigüis 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adolf Gogoll 4 Department of Chemistry-BMC, Uppsala University , BMC, Box 576, 75123, Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jon Jerlström-Hultqvist 3 Department of Medical Biochemistry and Microbiology, Uppsala University , BMC, Box 582, 751 23 Uppsala, Sweden 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Johan Åqvist 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dan I. Andersson 3 Department of Medical Biochemistry and Microbiology, Uppsala University , BMC, Box 582, 751 23 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dan I. Andersson Maria Selmer 1 Department of Cell and Molecular Biology, Uppsala University , BMC, Box 596, 751 24 Uppsala, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maria Selmer For correspondence: maria.selmer{at}icm.uu.se Abstract Full Text Info/History Metrics Preview PDF Abstract The first SAM degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as an S-adenosyl-L-methionine (SAM) hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha-beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography and NMR spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis, in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism. Introduction S-adenosyl methionine (SAM) plays many important roles in biology. It is an essential methyl donor for methyltransferases that act on nucleic acids, proteins, lipids and small molecules, but is also involved in many other reactions, e.g. as a substrate used in biosynthesis of polyamines and quorum sensing molecules (reviewed in ( Loenen, 2006 )). It is also involved in epigenetic changes in many organisms ( Janke et al., 2015 ; Su et al., 2016 ) and in bacterial restriction-modification systems where methylation of DNA is used to distinguish foreign DNA from host DNA ( Wilson and Murray, 1991 ). In early studies of nucleic acid methylation, it was observed that while infection of E. coli with phage T1, T2 and T4 induced higher levels of DNA methylation, infection with phage T3 reduced the degree of methylation of not only DNA, but also tRNA and rRNA ( Gold and Hurwitz, 1964 ). The reduction of methylation by T3 infection was associated with an immediate and dramatic lowering of the level of S-adenosyl methionine (SAM) in the E. coli cell extract. The degradation products were, using paper chromatography and chemical tests, identified as 5’-methyl-thioadenosine (MTA) and L-homoserine ( Gold and Hurwitz, 1964 ) ( Fig. 1 , top), which led to the claimed discovery of a potent T3-encoded SAM hydrolase enzyme ( Hausmann, 1967 ). Download figure Open in new tab Figure 1. SAMase reaction. Top: Hypothetical SAM hydrolase reaction previously suggested to be catalyzed by T3 SAMase. Bottom, green: SAM lyase reaction shown in this study to be catalysed by all tested bacteriophage SAMases. Subsequent work showed that the T3 SAMase was produced early in infection ( Gefter et al., 1966 ), encoded in the early transcribed portion of the phage genome ( Herrlich and Schweiger, 1970 ) and important for the counter defense of the bacteriophage against the type I restriction modification (RM) system of the host bacterium. In this type of RM system, SAM is an essential cofactor for both methylation of host DNA and restriction of target sequences in the foreign DNA. Two potential mechanisms for how the RM system could be impaired were presented. First, the lowered SAM levels prevented methylation of the host genome and the SAM-dependent restriction of the phage genome ( Krueger et al., 1975 ). Second, inhibition was observed to be independent of SAM degradation and possibly linked to an interaction with the restriction enzyme ( Spoerel et al., 1979 ; Studier and Movva, 1976 ). Recently, three additional SAM degrading enzymes were identified in a screen for bacteriophage DNA that could rescue an auxotrophic E. coli mutant where the isoleucine biosynthetic ilvA gene was deleted. Investigations using proteomics and RNA-seq showed that the phage-encoded polypeptides induced up-regulation of the biosynthetic pathway for methionine by degradation of SAM ( Jerlström Hultqvist et al., 2018 ), which together with the repressor protein MetJ acts as a co-repressor of the met regulon ( Weissbach and Brot, 1991 ). Isoleucine biosynthesis was rescued through a promiscuous activity of one of the up-regulated enzymes, MetB ( Fig. S1 ). One of the newly identified SAM degrading enzymes, Svi3-3, was cloned, expressed and purified. In vitro activity assays demonstrated that Svi3-3 catalyzed conversion of SAM to MTA. We herein present the first structure of a phage-encoded SAMase and explore the reaction mechanism using both computational and experimental biochemistry. Strikingly, the results unambiguously show that the phage-encoded SAMases are not hydrolases, as believed since the 1960s, but lyases ( Fig. 1 ). Results Structure determination of a SAM hydrolase enzyme The Svi3-3 enzyme was originally expressed from a library of fragmented environmental phage DNA. For this reason, the exact size of the original open reading frame was unknown, but an N-terminally hexahistidine-tagged 162 amino acid construct including some vector-derived sequence was shown to have SAMase activity ( Jerlström Hultqvist et al., 2018 ). The phage-derived sequence was subcloned to allow proteolytic removal of the hexahistidine tag, and based on predictions of a disordered N terminus, truncated variants were made. Full-length and N-terminally truncated variants of Svi3-3 were expressed with an N-terminal hexahistidine tag and purified on a Ni-column for structural studies. When expressed from the T7-promoter expression plasmid, the proteins were highly toxic and the tightly regulated, arabinose-inducible BL21 (AI) cells had to be used to allow cell growth until induction. An N-terminally truncated 146 amino acid construct of Svi3-3, where the hexahistidine tag had been removed ( Table S1 ), formed crystals in presence of SAM or the analogue S-adenosyl homocysteine (SAH). We here number its sequence starting at the N terminus of the TEV-cleaved protein, corresponding to an offset of −16 in relation to previous work ( Jerlström Hultqvist et al., 2018 ). The crystals diffracted to 1.45 Å resolution in space group F4 1 32 and the structure was solved using ab initio methods with Arcimboldo ( Rodríguez et al., 2009 ) ( Fig. 2A , F). There is one monomer in the asymmetric unit, forming a trimer around the 3-fold crystallographic axis. Apart from the disordered C-terminal 16 residues, the full structure could be built. Download figure Open in new tab Figure 2. Structure of SAM hydrolase Svi3-3. A. Structure of the Svi3-3 trimer in complex with SAH. B. Monomer structure of Svi3-3. C. Interactions of SAH (yellow sticks) at the trimer interface, D. Fo-Fc omit map for SAH contoured at 3 sigma. E. MTA (yellow sticks) and Pro (salmon sticks), overlay of SAH is shown in transparent grey sticks. F. Unbiased Arcimboldo ( Rodríguez et al., 2009 ) electron density map for MTA bound to Svi3-3, contoured at 2 sigma (0.39 e - /Å 3 ). Structure of Svi3-3 Svi3-3 forms a trimer of ferredoxin-like fold alpha-beta sandwiches, where each subunit has two helices on the outside and a five-stranded anti-parallel beta sheet at the center ( Fig. 2A-B ). The subunits pack in a triangular manner, forming continuous beta sheets with their neighbors and are each in a “velcro-like” topology where residue 2-6 at the N-terminus of one subunit form beta-sheet interactions with residue 120-124 in β8 of another subunit ( Fig. 2A ). The long β5 forms beta-sheet interactions with both of the other monomers through interactions between the backbones around residue 51 and 55. In addition, the end of the β5-β6 hairpin packs against α 1 of another monomer. The trimer interfaces are each stabilized by a single salt bridge, numerous hydrogen bonds and by hydrophobic interactions at the center. The substrate analogue SAH binds at the interface between two subunits ( Fig. 2C-D ), enclosed by the β5-β6 hairpin at the edge of the sheet. The adenosine base is recognized by interactions with the backbone carbonyl and amide of Ile77 and the side chain of Ser50. The ribose forms interactions with Ser50 from one subunit and Glu69 and Ser71 from the other subunit. The homoserine moiety forms interactions with backbone groups and the side chains of Glu69 and Gln104. In the structure from co-crystallization with SAM, catalysis has occurred and the product MTA is observed in identical position as the corresponding part of SAH, with the methyl group pointing away from Glu69 ( Fig. 2E-F ), while a proline molecule from the cryo buffer is bound in the second half of the active site, mimicking the second product. The protein structures are virtually identical in presence of SAH and MTA (root mean square deviation (RMSD) of 0.16 Å over 129 C α atoms). To test if the structure contains all the elements needed for activity in vivo , and because the start and end of the native phage protein sequence remains unknown, constructs truncated to only contain the ordered parts were tested for their ability to rescue the ilvA knockout mutant. Indeed, the N-terminally truncated construct additionally lacking the disordered C-terminus provided similar rescue to full-length Svi3-3, indicating that neither tail is needed for SAMase activity (data not shown). Based on this result, all further experiments were performed with the 146 amino acid Svi3-3 construct ( Table S1 ). Crystallization in absence of ligands led to a lower-resolution apo structure. The structure of Svi3-3 in apo state is overall very similar to the complex structures (RMSD of 0.84 Å over 124 C α atoms), but there is a shift of the N-terminus, a conformational change of residues 7-9 to form a more extensive interaction with β9 and the preceding loop of the neighboring subunit, leading to a slight loosening of the trimer ( Fig. 3 ). In addition, residues 64-65 in the β7-β8 hairpin are disordered. Download figure Open in new tab Figure 3. Comparison of the apo structure of Svi3-3 (yellow) and the MTA complex structure (colored as in A, transparent). Arrows indicate conformational differences between the N-terminal regions in the two structures. Small angle X-ray scattering (SAXS) data confirmed that Svi3-3 has a similar trimeric structure in apo state as in presence of SAM ( Fig. S2 ), indicating that product release and substrate binding does not require trimer dissociation. Similar structures A search for structures with similar fold and connectivity as Svi3-3 using PDBeFold ( Krissinel and Henrick, 2004 ) showed that bacterial PII signaling proteins and cation tolerance proteins of CutA type (belonging to a PII-like family) in the GlnB-like superfamily have similar trimeric architectures of ferredoxin-like folds. The closest structural neighbors superpose with RMSDs above 2 Å over no more than 86 C α atoms. PII regulatory proteins bind ATP/ADP and 2-oxoglutarate at the trimer interface, and conformational changes of loops in response to ligand binding and modification modulates binding to other proteins ( Forchhammer and Lüddecke, 2016 ). Intriguingly, the position of ATP is similar to where SAH binds to Svi3-3, although the binding site appears non-conserved. Catalytic residues The reaction mechanism of the presumed SAM hydrolases ( Fig. 1 ) is previously unexplored. Two possibilities would either be that the enzyme provides a catalytic base that by deprotonation activates a water molecule for nucleophilic attack on the γ carbon of SAM, or that a catalytic residue from the enzyme acts as nucleophile and attacks the γ carbon, forming a covalent intermediate with the substrate that subsequently gets hydrolyzed by reaction with a water molecule. In both scenarios, a catalytic amino acid in proximity to the site of hydrolysis would be needed. Inspection of the active site shows that Glu69 is relatively close to the site of cleavage ( Fig. 2C ), making this residue the most likely candidate. Another possible residue that could act as a base or a nucleophile is Tyr58, which in such case would need to donate a proton to the nearby Glu105 ( Fig. 2C ). To test these possibilities, Svi3-3 mutants Y58F, E69Q, E69A and E105Q were constructed, expressed and purified for in vitro activity assays. The Y58F mutant eluted mainly as monomer, indicating a de-stabilized trimer, while all other mutants migrated as trimers. The activity of wild type and mutant Svi3-3 variants in conversion of SAM to MTA was determined at 1 mM substrate concentration. The wild type truncated Svi3-3 construct showed an average turnover of 9.5 s -1 at 25°C, nearly doubled compared to the full length, His-tagged construct ( Jerlström Hultqvist et al., 2018 ), but with significant deviation between batches ( Fig. S3 ). The mutant enzymes all showed reduced activity compared to wildtype ( Fig. 4A , Fig. S3 ). For the Y58F and E105Q mutants, activity was 5-fold and 3-fold reduced, whereas for the E69Q and E69A mutants, activity was nearly abolished (4500- and 1500-fold reduction). Thus, Glu69 is in vitro critical for substrate binding, catalysis or both. Download figure Open in new tab Figure 4 Svi3-3 assays. A. Relative enzymatic activity of Svi3-3 variants. The data points are based on technical duplicates from two different protein purifications, where activity is related to WT purified at the same time. Absolute activity data is presented in figure S3. B. DSF data for Svi3-3 variants with different concentrations of SAM and SAH. Error bars correspond to +/-one standard deviation based on triplicate data. Thermal shift binding assays To characterize the binding of substrate and product to Svi3-3, thermal unfolding experiments were performed using differential scanning fluorimetry (DSF) ( Niesen et al., 2007 ). In presence of the substrate SAM, its analogue SAH or the reaction product MTA, the melting temperature of the wild type enzyme increased by up to 30°C, demonstrating a major stabilizing effect on the structure ( Fig. 4B ). Under the assay conditions, the substrate is most likely turned over, and as a result, the sample with SAM is stabilized by binding of MTA. A large stabilization was also seen in thermal denaturation assays followed by circular dichroism in presence of MTA (data not shown). The DSF assay clearly demonstrates that the E69Q mutant is not impaired in binding of SAM or MTA, but that binding of SAH is abolished ( Fig. 4B ). However, despite extensive trials, a structure of Svi3-3 E69Q with SAM could not be obtained. A crystal structure of Svi3-3 E69Q with MTA shows no major conformational changes of the enzyme, but a minor shift of the mutated side chain close to the reaction site (data not shown). Structure-guided sequence alignment supports conservation of structure and reaction mechanism A structure-guided multiple sequence alignment of Svi3-3 with T3 SAMase and two other polypeptides annotated with SAM hydrolase activity (Orf1 and Svi3-7 ( Jerlström Hultqvist et al., 2018 )) but with barely detectable sequence similarity, suggests that a similar core fold can be formed by all aligned enzymes ( Fig. 5 ). Only three amino acids are strictly conserved, and only a handful conservatively substituted. Based on the SAH structure, all the conserved residues are important for binding of SAM. Gly56 packs against the ribose, not allowing space for a side chain, Glu69 and Gln103 as described above form hydrogen bonds to the ligand and Tyr58 packs against the γ carbon and forms a hydrogen bond to Glu104, enclosing the ligand. Download figure Open in new tab Figure 5. Structure-guided sequence alignment of SAMases with demonstrated activity ( Jerlström Hultqvist et al., 2018 ). Secondary structure of Svi3-3 is displayed above the alignment. Red boxes with white letters indicate conserved residues and red letters in white boxes show conservatively substituted residues. Figure was prepared using ESPript ( Gouet et al., 2003 ). The Svi3-3 structure allows interpretation of the effect of loss-of-function mutations identified in the previous complementation studies ( Jerlström Hultqvist et al., 2018 ). One group of disabling mutations would act to sterically disturb the interaction between the monomers and/or change the character of the interface (R14C, V33D, V52D, E110K, G115V). Mutation of the conserved glycine (G56D) would clash with the ribose in MTA and SAH, and thereby disturb substrate binding. The remaining mutations (A13V, A93G, G95D) are likely to affect folding or stability of the structure. Mutagenesis of catalytic residues in homologous enzymes To validate the structure-guided sequence alignment, Glu68 in T3 SAMase, predicted by the sequence alignment ( Fig. 5 ) to be the equivalent of Glu69 in Svi3-3, was mutagenized and tested for in vitro activity. The E68Q mutant of T3 SAMase retained 20-30% activity compared to WT. In addition, mutant variants carried on an inducible plasmid were tested for their ability to rescue the ΔilvA auxotrophic mutant ( Fig. S1 ) ( Jerlström Hultqvist et al., 2018 ) under uninduced and induced conditions. Wild-type T3 SAMase because of its toxicity at higher expression level only shows rescue under uninduced conditions ( Jerlström Hultqvist et al., 2018 ). Both T3 SAMase and Orf1 contain two consecutive acidic residues (E67 and E68 in T3 SAMase, E50 and D51 in Orf1, Fig. 5 ), both of which were conservatively mutated. No rescue was observed either for the Svi3-3 E69Q, Svi3-3 E69A, Orf1 E50Q, or the T3 SAMase E68Q mutants, suggesting a loss or reduced activity of these variants ( Table S2 ) and validating the sequence alignment. Mutations of the neighboring acidic residues (E67Q in T3 SAMase and D51N in Orf1) still allowed rescue under the same conditions as the corresponding wild-type. Given that we observed measurable activity for the T3 SAMase E68Q mutant in vitro , we decided to insert different variants of the T3 SAMase on the chromosome, to allow titration of the expression levels. To this end, the different mutant gene variants were placed downstream of the p BAD promoter in a ΔilvA knockout mutant. Different concentrations of L-arabinose were used to induce the expression of these variants to determine if the variants could show in vivo activity at higher expression levels. For the E68Q mutant, growth was only observed at the highest concentration of arabinose (0.1%), where low enzymatic activity can be compensated by increased enzyme levels ( Table S3 ). Molecular dynamics simulations of Svi3-3 in complex with substrate To gain further insights into substrate binding to Svi3-3, molecular dynamics (MD) simulations were performed. An initial complex of Svi3-3 with SAM was generated from docking calculations, which indicated a slight binding preference for SAM over SAH. A total of 100 ns of MD simulation was run for both apo and holo Svi3-3. The holo simulation revealed that the SAM conformation in the active site is very stable, particularly the methionine part ( Fig. S4 ). In agreement with the experimental structure with SAH, the carboxylate group is strongly stabilized by the backbone amide hydrogens of Glu105 and Ser106, whereas the amino group is primarily stabilized by the side-chains of Glu69 and Gln104 ( Fig. S4A ). Moreover, the positively charged methionine S atom is consistently stabilized through a π-cation interaction with the Tyr58 side-chain ( Fig. S5A ). The adenosine part of SAM is primarily stabilized by H-bond interactions with the side-chain of Ser50 and the backbone of Val57 and Ile77, as illustrated in Figure S4 . When comparing the averaged root mean square fluctuations (RMSF) of the protein backbone, Svi3-3 was more flexible throughout the 100 ns MD simulation without SAM in the active site (apo). Here the average RMSF for the entire Svi3-3 trimer is 0.91 Å 2 and 0.86 Å 2 for the apo and holo, respectively. Interestingly, the residues surrounding the active site cavity, and in particular the methionine stabilizing residues 104 – 106, become significantly more flexible in the apo simulations ( Fig. S5B ). Thus, 100 ns of MD simulation demonstrated that Svi3-3 is most flexible in the regions surrounding the active site and that these regions become significantly more rigid upon substrate binding. Computational studies of the SAMase reaction mechanism The mechanism of enzymatic SAMase activity is previously unexplored. Thus, the observed active site conformations from the MD simulations were used to build a cluster model ( Fig. S5C ) to investigate the SAMase reaction mechanism with density functional theory (DFT). The simulations revealed one potential, but not optimally positioned, water molecule for hydrolysis that was stabilized by H-bonds between the backbone of Ser106 and Gln104, but there were no obvious residues within reach to activate this water by acting as a base. Glu69 was initially suspected to either work as a catalytic base, activating a water molecule for hydrolysis, or as a nucleophile, attacking the γ-carbon of SAM. However, no water molecule was observed with a suitable position with respect to Glu69 in the experimental structures or after the MD simulation, indicating that the role as a catalytic base is unlikely. Moreover, the observed SAM configuration relative to Glu69 was not optimal for attack on the γ-carbon of SAM. DFT geometry optimizations furthermore failed to locate any stationary point (transition state) for the attack, suggesting that the role as a nucleophile is also unlikely. Thus, it seems that the main role of Glu69 is to bind and orient the substrate in the active site by accepting H-bonds primarily from the amino group of SAM, but also from the 3’-hydroxyl group of the ribose ring ( Fig. S5C ). In fact, the only residue in the active site oriented properly for a potential hydrolase reaction mechanism is Tyr58. However, DFT calculations could exclude the possibility of Tyr58 acting as a nucleophile in such a mechanism. Further DFT geometry optimization, however, revealed a completely different mechanism, a unimolecular reaction resulting in the formation of homoserine lactone ( Fig. 6A ). The DFT optimized stationary points shown in Figure 6A indicate that Tyr58 is deprotonated and bridged by a water molecule to the protonated Glu105. Deprotonation of Tyr58 enhances its cation-π interaction with the S atom of SAM and the electrostatic preorganization weakens the bond to the ribose ring. Together with the amino group interaction with Glu69 this makes the configuration of SAM in the active site of Svi3-3 susceptible to intramolecular carboxylate oxygen attack on the γ-carbon. The activation energy for the formation of homoserine lactone in the DFT cluster model was calculated to be 16.8 kcal/mol with an exothermic reaction energy of − 5.7 kcal/mol ( Fig 6B ). Interestingly, in the product state ( Fig 6A ), Glu69 shares a proton with the SAM amino group and the water molecule also makes a short strong hydrogen bond with Tyr58. This water appeared to be a good candidate for nucleophilic attack on the carbonyl carbon of homoserine lactone, thereby forming a tetrahedral intermediate which could break down to homoserine. The calculated barrier for this step is 17.5 kcal/mol, which is not unrealistically high, but the reaction energy for the tetrahedral intermediate is 25.2 kcal/mol relative to the homoserine lactone ( Fig. 6B ). Thus, the DFT calculations indicate that Svi3-3 forms homoserine lactone, but that further breakdown to homoserine does not occur in the active site. Download figure Open in new tab Figure 6. DFT calculations on the SAM lyase reaction of Svi3-3. A. Optimized DFT structures for the reactant (left), transition (middle) and product (right) state in the SAM lyase reaction mechanism. B. Calculated free energy profiles for reactant (1), transition state (1-2) and product state (2) for the SAM lyase reaction mechanism. Further conversion from homoserine lactone (2) to the tetrahedral intermediate (3) that would form homoserine is not supported by the high DFT energies. Most importantly, the DFT calculations clearly predict that Svi3-3 is not a SAM hydrolase, but rather a SAM lyase, catalyzing the unimolecular transformation of SAM to homoserine lactone, whereafter this product is released from the active site. The carboxyl group of SAM appears essential for MTA production One prediction from the suggested reaction mechanism is that the carboxyl group of SAM is essential for the degradation of SAM by Svi3-3. To test this hypothesis, decarboxylated SAM (dcSAM) was produced enzymatically from SAM using SAM decarboxylase ( Cohn et al., 1983 ) and used as a substrate in the MTA formation assay. In support of the proposed mechanism, we observed no Svi3-3-catalysed production of MTA from dcSAM ( Fig. S6 ). In DSF binding assays, dcSAM does not induce a thermal stabilization of Svi3-3, indicating that the carboxyl group is important for binding and/or stabilization of the structure. Experimental verification of reaction products by thin-layer chromatography and NMR spectroscopy Our activity assay ( Jerlström Hultqvist et al., 2018 ) is based on detection of MTA that is also observed in the structure from co-crystallization of Svi3-3 with SAM. Thus, MTA is indeed formed in the reaction. In order to test the predictions from the DFT calculations, we used thin-layer chromatography (TLC), commonly used for separation of amino acids, to determine whether homoserine was formed. The substrate SAM and the products could be separated by TLC, the adenosyl-containing compounds were visualized under UV light and the amines were stained with ninhydrin after pre-treatment with β-mercapto ethanol (BME). Interestingly, we observed a reaction product with mobility and color upon staining distinct from homoserine, proving that Svi3-3 is not a SAM hydrolase. The same assay was performed with T3 SAMase and Orf1 and comparison with reference samples shows that for all three enzymes, a reaction product is observed that on TLC migrates and stains similarly to homoserine lactone ( Fig. 7A ), supporting the hypothesis that Svi3-3, T3 SAMase and Orf1 are indeed SAM lyases. Download figure Open in new tab Figure 7. Characterization of SAMase reaction products. A. TLC separation of enzymatic reactions and controls, shown in color and gray scale for clarity. (1: Svi3-3, 2: T3 SAMase, 3: Orf1, 4: SAM (17 nmol), 5: L-homoserine (20 nmol), 6: L-homoserine lactone (400 nmol)). B. 1 H NMR spectra at 600 MHz in sodium phosphate buffer, D 2 O, pH 7.4. Top: Enzymatic degradation of SAM (4 mM) (500 nM enzyme) after 45 min. Middle: Homoserine lactone (59 mM) showing onset of hydrolysis after 10 min. Bottom: Homoserine (57 mM). To confirm the identity of the reaction product, the enzymatic reaction products were examined with 1 H NMR and the spectrum compared to reference spectra for SAM, homoserine and homoserine lactone ( Fig. 7B , S7). The results confirm that homoserine lactone is formed on the same time scale as MTA. Upon incubation of homoserine lactone in phosphate buffer at pH 7.4, the lactone is transformed to homoserine through spontaneous hydrolysis ( Fig. S8 ). Discussion Relation to other enzymes with similar activity and structure Svi3-3 represents the first structure of a phage-encoded SAM degrading enzyme. There is no previously established reaction mechanism for these enzymes, and to our knowledge there are only two previous examples of enzymes that can cleave a trialkyl sulfonium substrate. A distinct type of SAM lyase, 1-aminocyclopropane-1-carboxylate synthase (ACC synthase, EC 4.4.1.14) exists in higher plants and some fungi. The products of this enzyme are MTA and 1-aminocyclopropane-1-carboxylate that is used in the biosynthetic pathway for ethylene. The reaction is PLP-dependent and the enzyme is structurally and mechanistically unrelated to the phage encoded SAM lyases ( Capitani et al., 1999 ). Instead, Svi3-3 shows structural similarity to PII and PII-like proteins, many of which bind nucleotides or nucleotide-derived metabolites in the inter-subunit clefts. Despite very low levels of sequence identity within those families, they have been suggested to have arisen by divergent evolution from a common ancestor ( Forchhammer and Lüddecke, 2016 ). Based on this similarity, we tested binding of ATP, ADP and AMP to Svi3-3 by DSF, but detected no interaction. Still, these two families of enzymes may have a distant evolutionary relationship. Substrate binding Svi3-3 forms a trimer in solution both in absence and in presence of substrate ( Fig. S2 ). Thermal shift binding experiments showed a major stabilization of Svi3-3 upon binding of MTA or SAH ( Fig. 4B ). Comparisons of the apo and ligand-bound structures as well as MD simulations indicate that stabilization is caused by ordering of the β7-β8 hairpin and tightening of the trimer around the ligand. Results presented here show that E69 in Svi3-3 plays a critical role in the enzyme, and the failure in getting crystals of an E69Q mutant with SAM suggests that binding of SAM may be associated with conformational changes that are incompatible with crystal packing or with the crystallization conditions. The observation that the E69Q mutant binds SAM but does not bind SAH indicates that although SAH only lacks one methyl group compared to SAM, the complex structure of wildtype Svi3-3 with SAH may not fully mimic the substrate-bound state. Since MTA and SAH show a perfect overlap between the two structures ( Fig. 2D ), any such difference in binding mode between SAM and SAH is likely to involve the methionine end of the substrate, where MD indicates that Tyr58 forms a cation-π interaction with the positively charged sulfur. The reason for why the E69Q mutant does not bind to SAH may be that two important interactions are lacking; Q69 cannot accept two hydrogen bonds ( Fig. 2C ) and there is no positive charge on the sulfur that can participate in the cation-π interaction with Tyr58. Phage-encoded SAMases are lyases Prompted by DFT calculations producing high energy barriers for a hydrolysis reaction within the active site of Svi3-3, the TLC assays and NMR show unambiguously that Svi3-3 is a SAM lyase forming homoserine lactone and MTA ( Fig. 7 ). The lactone is spontaneously converted to homoserine in solution ( Wu et al., 1983 ) ( Fig. S8 , 8 ). Previous attempts to set up a coupled SAM hydrolase activity assay for Svi3-3 using homoserine dehydrogenase or homoserine kinase failed to show homoserine production with the same rate as MTA production (data not shown). The observed rates were 1000-fold lower, and we can now explain that this is due to the slow and un-catalyzed formation of homoserine from the homoserine lactone that is enzymatically formed. Computational work by others suggests that the non-enzymatic degradation of SAM to homoserine lactone and MTA is slowed down by the favorable interactions of the carboxylate group with water ( Lankau et al., 2017 ). Thus, Svi3-3 increases the reactivity of the carboxylate group by excluding water from the corresponding part of the active site, while stabilizing a reactive conformation of the substrate. For this unimolecular reaction mechanism ( Fig. 8A ), only very few strictly conserved residues are required, as illustrated in the multiple-sequence alignment ( Fig. 5 ). Both hydrogen bond acceptors of Glu69 seem critical for stabilization of the reactive state ( Fig. 8 ), and Svi3-3 E69Q has nearly abolished activity. However, the effect of the corresponding mutation in T3 SAMase is not quite as dramatic. Since the level of sequence identity between the two enzymes is low, there will be many differences in the active site, and additional interactions may contribute to stabilizing the reactive state for the same mechanism in T3 SAMase. Download figure Open in new tab Figure 8. Mechanism of SAM degradation. A. The first step is catalyzed by the SAM lyase, releasing the products homoserine lactone and MTA. Hydrogen bonds between the enzyme and the reacting part of SAM are shown as dashed lines. The two subunits contributing to one active site are indicated in green and blue. B. Homoserine lactone is spontaneously hydrolyzed to homoserine in solution. The only previously partly characterized phage SAMase comes from phage T3. It was identified based on its anti-restriction activity, but the mechanism was never fully clarified. Anti-restriction activity of the closely related T7 phage is based on the OCR protein that forms a structure that mimics B-form DNA and blocks DNA binding of EcoKI and other type I RM systems ( Walkinshaw et al., 2002 ). The first structure from the SAM lyase enzyme family that we present here clearly proves that they have no structural similarity to the OCR protein. Instead, our data shows that also the T3 SAMase is a lyase and not a hydrolase, but future studies are needed to elucidate whether these enzymes also have additional mechanisms of anti-restriction activity ( Spoerel et al., 1979 ). Around the same time as the SAM-degrading enzyme from bacteriophage T3 was discovered, the same enzymatic activity was also found in extract from bacteria ( Shapiro and Mather, 1958 ) and yeast (S. Mudd, 1959 ; S. H. Mudd, 1959 ). In both of these systems, the reaction products were described as MTA and γ-aminobutyro-lactone (homoserine lactone), and the conversion to homoserine was considered to be spontaneous. In contrast, the phage enzyme from T3 was early described as a SAM hydrolase, and referred to as such until this day. In the early literature, homoserine lactone was identified as an intermediate ( Gold et al., 1964 ) but, perhaps due to the available methods at the time, it was not realized that homoserine was formed on a different time-scale from MTA, indicating a spontaneous and not enzyme-catalyzed reaction. For this reason, it is not until now that we can correct the functional annotation to SAM lyase. The SAM lyases show very low sequence conservation and large variations in size ( Fig. 5 ), and future studies will elucidate the relationship between structure and activity in this family of enzymes, their prevalence and their exact biological roles in different organisms. Methods Cloning For expression of an N-terminally truncated construct of Svi3-3, the svi3-3 gene was PCR amplified using Pfu DNA polymerase with primers Svi3-3_d19f and Svi3-3_r1 and cloned into the pEXP5-NT/TOPO vector (Invitrogen) according to the manufacturers protocol. Transformants were selected on LA plates supplemented with 100 µg/ml ampicillin. The correctness of plasmid pEXP5-Svi3-3_d19 was confirmed by sequencing (Eurofins). The resulting plasmid encoded amino acid 20-162 of the original His-tagged Svi3-3 polypeptide (corresponding to residue 5-147 from the phage-encoded sequence) fused to an N-terminal hexahistidine tag followed by a TEV cleavage site. The sequence is numbered starting at the N terminus of the TEV-cleaved protein sequence, corresponding to an offset of −16 in relation to previous work ( Jerlström Hultqvist et al., 2018 ). Site-directed mutagenesis Svi3-3-d19 mutants Y58F, E69Q, E69A and E105Q were generated by site directed mutagenesis of pEXP5-Svi3-3_d19 using the QuickChange II protocol (Stratagene) using the primers listed in Table S4 . Mutations were confirmed by DNA sequencing. Protein expression and purification Expression plasmids were transformed into BL21-AI cells and plated on LA plates containing 50 µg/ml ampicillin and 0.1% glucose. For protein expression, 5 ml overnight culture in LB containing 50 µg/ml ampicillin and 0.1% glucose was used to inoculate 800 ml LB medium with the same composition and incubated at 37°C with shaking. When OD 600 reached 0.9, expression was induced with 0.2% L-arabinose and the culture was further incubated at 37°C for 4 hours before harvest by centrifugation. The cell pellet was resuspended in buffer A (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 5 mM BME) including cOmplete EDTA-free protease inhibitor (Roche) and subjected to lysis by sonication. After centrifugation at 30,000 x g for 30 min, the supernatant lysate was clarified by filtration through a 0.45-µm-syringe filter, loaded to a gravity column containing pre-equilibrated Ni-Sepharose (GE Healthcare) and incubated under slow rotation for 10 min at 4 °C. The column was washed extensively with buffer A supplemented with 20mM imidazole, and the His-tagged protein was eluted with buffer A containing 500 mM imidazole. Protein-containing fractions were loaded onto a HiLoad 16/60 Superdex 75 column equilibrated with buffer B (25 mM Tris-HCl, 150 mM NaCl, pH 8.0). Wildtype Svi3-3_d19, E69Q, E69A and E105Q mutants eluted as trimers, while the Y58F mutant eluted mainly as monomer. Peak fractions were pooled and concentrated to 2 mg/ml. To cleave off the His-tag, the protein was incubated at 4°C overnight with a 1:10 molar ratio of TEV SH protease ( Van Den Berg et al., 2006 ). The cleavage reaction was passed through Ni-Sepharose before being loaded onto a HiLoad 16/60 Superdex 75 column equilibrated in buffer B. Peak fractions were concentrated to 10 mg/ml for further use. T3 SAMase was produced by in vitro transcription-translation as previously described ( Jerlström Hultqvist et al., 2018 ). Crystallization, data collection and structure determination Crystallization was done using the sitting-drop vapour diffusion method at room temperature (293 K). Crystals grew in 2-10 days in drops containing 1 µl Svi3-3_d19 (10 mg/ml, with or without 5 mM SAH/SAM) and 1 µl of reservoir solution containing 0.4-0.6 M ammonium phosphate. Crystals were cryo-protected in reservoir solution supplemented with 1.5 M proline and vitrified in liquid nitrogen for data collection. All data were collected at ESRF beamline ID23-1 at 100K and processed with XDS( Kabsch, 2010 ). The Svi3-3_d19 structure with SAM was solved with ab initio methods using Arcimboldo_lite( Rodríguez et al., 2009 ) run on the National Supercomputer Center (NSC) in Linköping and a 15 amino acid helix as search model( McCoy et al., 2007 ). The structure was manually rebuilt in Coot ( Emsley et al., 2010 ) and refined using phenix.refine( Afonine et al., 2012 ). Statistics for data collection and refinement are summarized in Table 1 . View this table: View inline View popup Download powerpoint Table 1. SAXS SEC-SAXS data for Svi3-3_d19 samples were collected at the Diamond Light Source on beamline B21. In-line SEC-SAXS was performed using an Agilent 1200 HPLC system connected Shodex KW403 column. Data were recorded on a Pilatus 2M detector with a fixed camera length of 4.014 m and 12.4 keV energy allowing the collection of the angular range q between 0.0038–0.42 Å -1 . His 6 -tagged Svi3-3_d19 samples at 10-13 mg/ml concentration with and without 5 mM SAM were loaded onto the size exclusion column previously equilibrated in 25 mM Tris-HCl pH 8.0, 150 mM NaCl. The data was initially subtracted with the buffer and data processing was performed using ScÅtter ( Förster et al., 2010 ). Further data analysis was performed with Primus ( Konarev et al., 2003 ) and SAXSMoW ( Piiadov et al., 2019 ). Activity assay The activity of wildtype and mutant versions of the N-terminally truncated Svi3-3 construct was determined according to the previously published discontinuous assay ( Jerlström Hultqvist et al., 2018 ), by separation of SAM and MTA using cation exchange chromatography. All experiments were conducted at 25°C and the enzyme concentration and duration of the experiment were adjusted to the level of activity, ranging from 50 to 500 nM enzyme and 10 min to 1-week incubation. The experiments were done in biological duplicates (two separately purified batches of each protein) and technical duplicates (two independently pipetted and measured enzymatic reactions). Differential scanning fluorimetry The protocol was adopted from Niesen et al.( Niesen et al., 2007 ). Each 25 µl reaction consisted of 20 µM wild type or mutant Svi3-3 in 25 mM HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), 150 mM NaCl, 0.2 µl 50x SYPRO orange dye and 0 - 2500 µM of SAM or SAH. Reactions were done in technical triplicates in a BioRad CFx connect real-time system and subjected to a temperature gradient from 15 °C to 95 °C with an increment of 0.2 °C per 30 s. Structure-guided sequence alignment Structure-guided sequence alignment was performed with PROMALS3D ( Pei et al., 2008 ) and manually edited. The sequence alignment figure was prepared using ESPript( Gouet et al., 2003 ). Inserting different gene variants encoding T3 SAMases on the chromosome The different gene variants encoding the wild type and mutant T3 SAMase were inserted on the chromosome of an ΔilvA auxotrophic mutant of E.coli K-12 MG1655 by λ-red recombineering as previously described ( Datsenko and Wanner, 2000 ). Each of the variants was used to replace the araBAD operon, so that the expression for these was under the control of the p BAD , the native promoter for the araBAD operon. Briefly, the first step involved replacing the araBAD operon in an ΔilvA E. coli K-12 MG1655 strain by cat-sacB-yfp cassette. The cassette was PCR amplified using the primers araBAD_cat_sacB_F and araBAD_cat_sacB_R ( Table S4 ). Native T3 SAMase, and variants with mutations at E67Q and E68Q were PCR amplified using specific primers that contained homologies surrounding the araBAD operon at the 5’ end followed by sequences that allowed amplification of the T3 SAMase variants from the respective plasmids (ara_t3samF, ara_t3samR). PCR products were purified, DNA was transformed into the strain containing the cat-sacB-yfp cassette at the araBAD location, and transformants were selected on sucrose plates. Variants were confirmed by Sanger sequencing (using test_primer_F, test_primer_R, Table S4 ). In vivo complementation of ΔilvA mutant with different variants of Svi3-3, Orf1 and T3 SAM hydrolase variants Two different approaches were used to determine if the different variants of Svi3-3, Orf1 and T3 SAMase could complement the ΔilvA auxotrophic mutant. In cases where the variant was present on a plasmid, the plasmid was transformed into the ΔilvA auxotrophic mutant and was selected on LA-ampicillin (50 µg/ml) or LA-chloramphenicol (15 µg/ml) plates. The transformants were then re-streaked, and the re-streaked colonies were tested for growth on M9-Glucose (0.2%) minimal media plates, with or without IPTG. On each test plate, the ΔilvA auxotrophic mutant containing the empty vector was used as a negative control. The same approach was used to test functionality of the T3 SAMase variants that were present on the chromosome, with induction being obtained using different concentrations of L-arabinose (0%, 0.01%, 0.05% and 0.1%). In vivo rescue assay E69A and E69Q variants were constructed for full-length Svi3-3 and Svi3-3_d19 to test for their ability to rescue the ΔilvA mutant ( Jerlström Hultqvist et al., 2018 ). Synthetic genes (Eurofins) containing the desired mutations and Kpn 1 and Xba 1 cleavage sites were cleaved-out of the original vector and ligated into a modified version of the pCA24N plasmid, purified on a spin-column and transformed directly into the ΔilvA mutant. Plasmids were extracted from isolated colonies and sequenced to confirm the correct sequence. The respective clones were then checked for their ability to grow on minimal glucose plates. All in vivo complementation experiments were done in biological duplicates. Molecular Dynamics Molecular dynamics (MD) simulations of the SAM hydrolase trimer with (holo) and without substrates (apo) in the active sites were performed with Desmond ( Bowers et al., 2006 ; Schrödinger, 2018 ) using the OPLS3 force field ( Harder et al., 2016 ; Jorgensen et al., 1996 ; Jorgensen and Tirado-Rives, 1988 ; Shivakumar et al., 2010 ). The Svi3-3 crystal structure in complex with SAH was used as starting conformation for the simulations. In the apo simulations the native substrate, S-adenosyl-methionine (SAM), was docked to the active site using Glide ( Friesner et al., 2006 , 2004 ; Halgren et al., 2004 ; Schrödinger, 2018 ). The docking grid was generated with the OPLS3 force field centered on the S-adenosyl homocysteine (SAH) inhibitor bound to the active site of the Svi3-3 crystal structure. The maestro system builder (Schrödinger, n.d.) was used to solvate the SAM hydrolase trimer with TIP3P ( Jorgensen et al., 1983 ) water molecules in an orthorhombic box with buffer distances of 10 Å to the boundary on all sides. The system was neutralized by addition of Na+ ions and the final simulation box consisted of 60972 atoms. A total of 100 ns MD simulation at 298 K was run in the NPT ensemble using the reference system propagator algorithm (RESPA) time stepping scheme ( Tuckerman et al., 1991 ) with time steps of 2 fs for bonded terms, 2 fs for van der Waals and short-range electrostatic interactions and 6 fs for long-range electrostatic interactions. Short range Coulomb interactions were treated with a cutoff radius of 9 Å. Long-range interactions were treated with the smooth Particle Mesh Ewald method ( Darden et al., 1993 ) with a tolerance of 10−9. The NPT ensemble was calculated with the Nose-Hoover chain thermostat method ( Hoover, 1985 ; Nosé, 2002 ), using a relaxation time of 1 ps, and the Martyna-Tobias-Klein barostat method ( Martyna et al., 1994 ), using isotropic coupling with a relaxation time of 2 ps. Computational reaction mechanism investigations The catalytic mechanism in SAM was investigated with density functional theory (DFT) calculations. A cluster model ( Fig. 5A ) was generated from a snapshot of the equilibrated Svi3-3 X-ray structure taken from the MD simulation described above. The active site model was composed of the backbone atoms of Val17, Gly18, Leu19, Asn20 and Val21 chopped at the N- and C-terminal, Tyr58 and Glu69 chopped at the CA position and Gln104, Glu105 and Ser106 (with backbone) chopped at the N- and C-terminals. A smaller substrate mimicking S-adenosyl methionine (SAM) was used for the DFT calculations. Here adenosine was deleted from SAM, resulting in 2-ammonio-4-((R)-ethyl(methyl)sulfonio)butanoate, or S-ethylmethionine (SEM). In addition, a total of 6 water molecules from the MD snapshot were included in the cluster model. To account for the steric effect of the surrounding parts of the protein, atoms in the chopped positions were kept fixed to their original positions ( Fig. 5A ). The final model after addition of methyl groups to the chopped protein positions consisted of 194 atoms (716 electrons). All DFT calculations were performed using the Gaussian 09 ( Frisch et al., 2009 ) package. Geometry optimizations and frequency calculations were computed with the B3LYP functional ( Becke, 1993 ) and the 6-31G(d,p) basis set. Dispersion effects were included in all calculations using Grimme’s B3LYP-D3 method ( Grimme et al., 2011 , 2010 ). Intrinsic reaction coordinate calculations were performed in both directions from the transition state to verify that the correct minima are connected. Solvent effects were obtained by single-point calculations on the optimized stationary points with the solvent model based on density (SMD) ( Marenich et al., 2009 ). Electronic energies were calculated from single-point calculations on the optimized geometries (RS, TS and PS) at the b3lyp/6-311G+(2d,2p) level of theory. The final reported energies are the electronic energies with the large basis set corrected for zero-point energies (ZPE) and solvent effects in kcal/mol. Production of SAM decarboxylase Cells from the ASKA library encoding the SAM decarboxylase (SDC) enzyme were inoculated in LB containing 34 µg/ml chloramphenicol. 5 ml of saturated culture was used to inoculate 800 ml LB medium with the same composition and incubated at 37 °C until OD 600 reached 0.6. Expression was induced with 0.5 mM IPTG and the culture was incubated overnight at 20 °C. The cells were harvested by centrifugation at 4 °C. The pellet was washed in 25 mM Tris pH 8, 150 mM NaCl, and pelleted again at 8 °C. The cells were resuspended in buffer A (50 mM Tris-HCl pH 8, 300 mM NaCl, 10 mM MgSO 4 , 5 mM BME) including cOmplete EDTA-free protease inhibitor (Roche) and subjected to lysis by sonication. After centrifugation at 30,000 x g for 30 min, the supernatant was clarified by filtration through a 0.45-µm-syringe filter, loaded in a gravity column containing 2 mL of pre-equilibrated Ni-sepharose (GE Healthcare), and incubated under slow rotation for 30 min at 8 °C. The column was washed extensively with buffer A supplemented with 20 mM imidazole, and the His-tagged protein was eluted with buffer A containing 500 mM imidazole. Protein-containing fractions were loaded onto a HiLoad 16/60 Superdex 200 column previously equilibrated with buffer B (25 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM MgSO 4 , 5 mM BME). Peak fractions were pooled and concentrated to 5 mg/ml. SAM decarboxylation 0.5 mM SAM was incubated with 20 µM SDC for 2 hours at 37 °C in reaction buffer (20 mM HEPES pH 7, 50 mM KCl, 10 mM MgSO 4 ). After the incubation, 40 µL of the reaction was quenched with the same volume of quenching buffer (50 mM citrate pH 2.6). The rest of the reaction was filtered through a 3 kDa cutoff concentrator to remove the enzyme. The flow-through was collected and dcSAM concentration was determined by the absorbance at 260 nm. The fraction of dcSAM was determined using ion exchange chromatography. dcSAM assay 0.32 mM dcSAM mix (72% dcSAM) was incubated with 0.1 µM Svi3-3 in reaction buffer at 37 °C. As a control, dcSAM without Svi3-3 was incubated for the same time. SAM (0.32 mM) was incubated with 0.1 µM Svi3-3 and as a control only SAM (0.32 mM) was incubated for the same period of time. Samples of 40 µL were quenched with the same volume of quenching buffer after 0.5 and 23 hours. Samples were analyzed with the same cation exchange method as for the standard activity assay ( Jerlström Hultqvist et al., 2018 ) but using a linear buffer gradient over 7 column volumes. TLC For TLC experiments, reactions containing 2 mM SAM and 0.20 μM Svi3-3, 0.20 μM Orf1 or 0.25 μM T3 SAMase in 50 mM NaPi pH 7.4 were incubated 20 min at 25°C. MTA product formation of >70% was verified using ion exchange chromatography. 5 x 2ul reaction was loaded on a TLC Silica gel 60 F 254 plate and developed using a mobile phase of 55 % n-butanol, 30 % H 2 O, 15 % acetic acid. The plate was treated with BME followed by ninhydrin staining ( Basak et al., 2005 ). Briefly, the dried plate was sprayed with 1 % BME in acetone, heated with a hair dryer, sprayed with 0.25 % ninhydrin in acetone and heated again until spots were clearly visible. NMR To analyze reaction products by NMR spectroscopy, a 700 µl reaction mixture was prepared containing 4 mM SAM and 500 nM Svi3-3 d19 in 100 mM Na phosphate buffer pH 7.4 and 90% D 2 O. The pH of the sample was adjusted to 7.4 after addition of SAM with 5M NaOH. The reaction was incubated for 20 min at 25 °C and shock-frozen in liquid N 2 and stored at - 80 °C until measurement. Turnover of >90% of substrate was verified by ion exchange chromatography. 1 H NMR spectra were recorded at 600.18 MHz on a Bruker Avance Neo spectrometer equipped with a TCI (CRPHe TR- 1 H & 19 F/ 13 C/ 15 N 5mm-EZ) cryogenic probe for samples in aqueous sodium phosphate buffer (NaPi, 100 mM, pH 7.4, solvent D 2 O) at 25°C. Typically 64 – 128 scans were accumulated with a relaxation delay of 0.7 s and an acquisition time of 2.75 s, using the zg30 pulse sequence. Spectra were obtained by zero filling the recorded 32k data points to 128k, followed by multiplication with an exponential weighting function and Fourier transformation. The formation of homoserine lactone in the enzymatic reaction was confirmed by comparison with the 1 H NMR spectrum of an authentic sample (from Sigma) dissolved in the same buffer, and by comparison with literature data( Helms et al., 1988 ). Homoserine lactone spectra were also recorded for various concentrations (70 mM, 17.5 mM, 4.4 mM, 1.1 mM, 0.3 mM) and for various times after sample preparation ( Fig. S6 ). The spectra indicated a gradual hydrolysis of the lactone to homoserine that was identified by comparison with literature 1 H NMR data ( Jamieson et al., 2009 ) and an authentic homoserine sample. Data availability The structure coordinates have been deposited to the wwPDB with accession codes 6ZM9 (Svi3-3 with MTA), 6ZMG (Svi3-3 with SAH) and 6ZNB (apo Svi3-3). SAXS data has been deposited in SASBDB with accession codes SASDJ65 (Svi3-3 with SAM) and SASDJ55 (apo Svi3-3). Supplemental material Download figure Open in new tab Figure S1. Proposed mechanism of rescue of an ilvA auxotrophic mutant by SAMases ( Jerlström Hultqvist et al., 2018 ). Expression of enzymes in the methionine synthesis pathway is repressed by MetJ in complex with SAM. When SAM is degraded by a SAMase, expression increases for the enzymes in the metJ regulon. At high expression level, the promiscuous function of MetB, to produce alpha-ketobutyrate from O-succinyl homoserine (green arrow), provides rescue of isoleucine biosynthesis. Download figure Open in new tab Figure S2. SEC-SAXS of Svi3-3-d19. A . Overlay of signal plots from SEC-SAXS of Svi3-3_d19 in presence and absence of 5 mM SAM (plotted with an offset of 0.05 for clarity). B. Zoom in of signal plot for Svi3-3_d19 apo. Black markers indicate the radius of gyration calculated from the individual scattering curves. Grey bars indicate the data frames used for further analysis. C. Zoom in of signal plot for Svi3-3_d19 in presence of SAM. Black markers indicate the radius of gyration calculated from the individual scattering curves. Grey bars indicate the data frames used for further analysis. D. Scattering curves for Svi3-3_d19 in presence and absence of SAM. Colors as in A. The blue curve is for clarity plotted with an offset of +1. E. Guinier plot for Svi3-3_d19 apo. F. Guinier plot for Svi3-3_d19 in presence of SAM. Download figure Open in new tab Figure S3. Enzymatic activity of Svi3-3 variants. Data is from technical duplicates from two different protein purifications (ordered from left to right) with the average represented by a black line. Calculated average activities +/-one standard deviation were: WT: 9.5 +/-1.7 s -1 , Y58F: 1.9 +/-0.7 s -1 , E69Q: 0.002 +/-0.0006 s -1 , E69A: 0.007 +/- 0.0016 s -1 , E105Q: 3.3 +/-0.99 s -1 Download figure Open in new tab Figure S4. Protein-ligand interactions obtained from 100 ns MD simulations illustrating the percentage of interaction from the total simulation time. Download figure Open in new tab Figure S5. Computational modeling of Svi3-3 in complex with SAM. A. Snapshot from 100 ns MD simulations illustrating the dominant interactions for the methionine part of SAM in the active site. B. Average backbone root mean square fluctuation (RMSF) of active site residues 104 – 106 averaged over 100 ns MD simulation apo and holo. C. DFT cluster model utilized for the DFT calculations. Atoms marked with * are kept fixed during the calculations. Download figure Open in new tab Figure S6. Ion exchange chromatography separation of dcSAM reactions and controls. A. 0.32 mM dcSAM mix (72 % dcSAM) incubated without enzyme B. 0.32 mM dcSAM mix (72 % dcSAM) incubated with 0.1 μM Svi3-3_d19 C. 0.32 mM SAM incubated without enzyme D. 0.32 mM SAM incubated with 0.1 μM Svi3-3_d19. Download figure Open in new tab Figure S7. 1 H NMR spectra of reference samples. A. Homoserine lactone (600 MHz, 59 mM in sodium phosphate buffer, D 2 O, pH 7.4). B. Homoserine (600 MHz, 57 mM in sodium phosphate buffer, D 2 O, pH 7.4). Download figure Open in new tab Figure S8. 1 H NMR spectra of a reference sample of homoserine lactone at various concentrations and exposure times to sodium phosphate buffer showing progressive hydrolysis to homoserine (600 MHz, sodium phosphate buffer, D2O, pH 7.4). Spectrum 1: 70 mM (t =0); spectrum 2: 17.5 mM (t = 50 min); spectrum 3: 4.4 mM (t =547 min); spectrum 4: 1.1 mM (t = 579 min); spectrum 5: 0.3 mM (t = 603 min). View this table: View inline View popup Download powerpoint Table S1: Sequences of Svi3-3 constructs in this study View this table: View inline View popup Download powerpoint Table S2: In vivo complementation assay using different variants of Svi3-3, Orf1 and T3 SAMase, present on the IPTG-inducible plasmids pCA24N –gfp (Svi3-3 and Orf1) and pRD2 (T3 SAMase). View this table: View inline View popup Download powerpoint Table S3: In vivo complementation assay using different variants of T3 SAMase, present on the chromosome View this table: View inline View popup Download powerpoint Table S4: Primer sequences Acknowledgements The authors want to thank Ulrika Yngve for advice regarding TLC. This work was supported by grants from the Knut and Alice Wallenberg foundation (Evolution of new genes and proteins) to MS, JÅ and DIA, from the Swedish Research Council grant 2017-03827 to MS, 2017-01527 to DIA and from the Research Council of Norway through a Centre of Excellence and project grants (Grant Nos. 262695 and 274858) to GVI. We are grateful for access to beamlines ID23-1 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France and beamline B21 at the Diamond light source, Didcot, UK. 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