Discovery and Biosynthesis of Nitrilobacillins by Post-translational Introduction of C-Terminal Nitrile Groups

Nitrile-containing natural products are produced in all kingdoms of life . Despite the wide application of nitrile -containing peptide scaffolds in medicinal chemistry , the presence of the nitrile group is unprecedented in ribosomally synthesized and post - translationally modified peptide s (RiPPs). In this work, we report the identification and characterization of a RiPP biosynthetic gene cluster (BGC) , where an asparagine synthetase-like (AS-like) protein encoded in the BGC converts the C-terminal carboxylate of the precursor peptide to a nitrile. Furthermore, a multinuclear nonheme iron-dependent oxidative enzyme (MNIO) and an α-ketoglutarate-dependent HExxH motif -containing enzyme (αKG-HExxH) perform stereoselective β -hydroxylation of aspartate and proline residues, respectively. The final product is a cysteine protease inhibitor and shows that Nature makes similar warheads as found in synthetic therapeutics such as the active .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 2 ingredient of Paxlovid. These findings extend our understanding of the structural and functional diversity of RiPPs.

Since the discovery of allyl cyanide in 1863,1 nitrile-containing natural products have been reported to be produced by animals, plants and microorganisms.2-5 The electrophilic nature of the nitrile carbon contributes to the activity of nitrile-containing metabolites in biology and medicinal chemistry.6-8 In nature, production of the nitrile group is achieved mostly via dehydration of the corresponding aldoximes catalyzed by various enzymes (Figure S1a ).9,10 Alternatively, nitrile formation proceed s through oxidative or ATP - dependent mechanisms (Figure S1a).5,11-15 Nitrile groups have been identified in diverse classes of natural products including glycosides, alkaloids, and terpenes, but nitrile- containing peptide natural products are rare, with auranthine the only example discovered thus far (Figure S1b). In contrast, use of the nitrile functional group is common in synthetic peptides designed to inhibit proteases, with one notable example being nirmatrelvir, a key ingredient in the SARS-CoV-2 therapeutic Paxlovid that inhibits the viral main protease.16 Recent estimates suggest that a very large fraction of natural products (up to 97%) remains to be discovered.17 Peptide secondary metabolites are mostly derived from non- ribosomal or ribosomal pathways. 18,19 Compared with non -ribosomal pathways , which include the biosynthesis of auranthine, ribosomally synthesized and post -translationally modified peptides (RiPPs) are differentiated by the presence of precursor peptides that are genetically encoded. Estimations of the abundance of various natural product classes in defined natural environments show that RiPPs are a major family ,20 and in some environments such as the human microbiome, RiPPs appears to be the most prevalent class of natural products. 21 Maturation of RiPPs involve s diverse post-translational modifications (PTMs) of the precursor peptide.22 Around 50 classes of RiPPs have been reported to date that are categorized by class -defining PTMs. The number of distinct PTMs continues to expand as a result of the rapid increase in genome sequences over the past two decades.23 A large number of different enzyme families have been linked to RiPP biosynthesis.24 Because the substrate sequences of RiPP modifying enzymes are .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 3 encoded within the BGC, f unctional characterization of RiPP biosynthetic enzymes for which activity has not been reported previously is less challenging compared with their counterparts in the biosynthe sis of other natural products . These features have made RiPP BGCs excellent repositories to discover novel enzyme chemistries.23 Recent studies have expanded the chemical space of the products of non-heme iron dependent enzymes in RiPP biosynthesis ,25-32 including RiPP BGCs that encode MNIO and αKG -HExxH proteins. In the current work, we focused on a BGC that contains members of both enzyme classes from Peribacillus simplex VanAntwerpen02. We demonstrate that the MNIO and HExxH enzymes catalyze β-hydroxylation of aspartate and proline residues, respectively. The more unique reaction in the pathway features an AS-like enzyme that unexpectedly installs a nitrile group at the C -terminus of the precursor peptide. We therefore termed the products from this BGC nitrilobacillins because orthologous pathways were only identified in Bacillus-related genera. Production of the nitrile-containing peptide seems to be regulated by two different mechanisms. First, nitrile installation requires Asp hydroxylation by the MNIO. Second , an apparent pseudo enzyme related to the MNIO protein is encoded within the BGC, which can compete with the MNIO protein in binding to the MNIO partner protein , thereby preventing nitrile biosynthesis. The RiPP products are inhibitors of several cysteine proteases tested. This work therefore expands RiPP biosynthe sis with a pharmaceutically significant pharmacophore.

Identification of the pes BGC During our genome mining efforts for novel RiPP BGCs encoding MNIOs, a BGC (pes cluster) from Peribacillus simplex VanAntwerpen02 was identified. The BGC encodes the αKG-HExxH protein PesO, two precursor peptides PesA1/A2, a n MNIO enzyme PesH, a putative MNIO partner protein PesI, a n AS-like enzyme PesC, a hypothetical protein PesX, two M16 peptidases PesP1/P2, and a transporter protein PesT (Figure 1a). Unlike previously reported MNIO enzymes which require a partner protein that is encoded adjacently within the BGC ,25,27,30,33-36 the putative MNIO partner protein .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 4 gene pesI is positioned next to pesX. Initial bioinformatic analysis of PesX suggested that it is a hypothetical protein from an unknown protein family, but structural analysis with AlphaFold 337 as well as a Foldseek38 search revealed that PesX likely possesses a triose-phosphate isomerase (TIM)-barrel fold like MNIOs and shares structural similarity to another MNIO protein MbnB (Figure S2). However, a sequence alignment of PesX with characterized MNIO proteins demonstrated that it lacks the iron-binding residues that are conserved in MNIOs (Figure S3).39 Therefore, the role and function of PesX was unclear. A sequence h omology search of the enzymes in the pes cluster using BLASTp, followed by genome neighborhood analysis using RODEO40 lead to the discovery of 26 gene clusters that resemble the pes cluster composition. These orthologous clusters all contain homologs of PesX, PesI, PesH and PesC. αKG-HExxH proteins are only present in ~ 75% of the clusters and when they are absent, they are replaced by a predicted arginase (e.g. Figure S4). Alignment of the precursor peptides from these homologous clusters reveals several conserved residues including a conserved C-terminal R(V/T)DN motif (Figure 1b). Notably, a proline residue before the R(V/T)DN motif only co-occurs when an αKG-HExxH protein is encoded in the BGC, hinting that the latter enzyme may modify this Pro residue. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 5 Figure 1. a) Composition of the pes BGC from Peribacillus simplex VanAntwerpen02 and the amino acid sequence of precursor peptides PesA1/2. The numbering is based on the C-terminal fragment obtained upon GluC-digestion; b) Sequence logo of precursor peptides from BGCs homologous to the pes BGC (4 2 total sequences, 21 unique sequences (Figure S5), identical sequences were removed). Characterization of the product of the pes BGC Because the native pes BGC encoding strains were not available to us , we investigated the functions of the encoded enzymes by heterologous expression in E. coli. A His 6-tag was appended to the N -terminus of the precursor peptides, and they were expressed separately or co -expressed with a subset of putative modifying enzymes. Following immobilized metal affinity chromatography (IMAC) purification, the purified peptides were digested with endoproteinase GluC and characterized by mass spectrometry. We co-expressed both precursor peptides PesA1 and PesA2 individually with the various enzymes encoded in the pes BGC with essentially the same results. We will describe the data with PesA2 here and refer to the Supporting Information for the analogous data for PesA1. Co-expression of PesA2 with the MNIO protein PesH and its potential partner PesI and analysis by liquid chromatography-high resolution mass spectrometry (LC-HRMS) revealed the emergence of a new species with a +16 Da mass shift with respect to the unmodified PesA2 (Figure 2). The site of PesHI modification was located to Asp21 by high-resolution tandem mass spectrometry (HR-MS/MS) (Figure S6). Marfey’s analysis was used to determine the position and stereochemistry of the .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 6 oxidation.41,42 After hydrolysis of the PesHI-modified peptide, the hydroxylated aspartate was reacted with 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (L-FDAA) and analyzed by liquid chromatography coupled to mass spectrometry detection ( LC-MS). The product coeluted with an L-threo-3-hydroxyaspartate standard that was derivatized in the same manner (Figure 3a and S7). Collectively, these observations demonstrate that PesHI facilitate stereoselective hydroxylation of Asp21. Next, we included the αKG -HExxH protein PesO in the co -expression system in addition to PesHI, for which we will use the designation PesOHI . LC-HRMS analysis of the endoproteinase GluC-digested product peptide PesA2-PesOHI revealed the dominant production of a M+32 Da species (Figure 2). This additional +16 Da mass shift compared to the PesHI modification was assigned to the Pro1 8 residue by HR-MS/MS analysis (Figure S6). We again performed Marfey’s analysis on the PesOHI -modified peptide to elucidate the identity of the hydroxyproline residue (Figure 3b and S7). Coelution with L-3S-hydroxyproline derivatized with the M arfey’s reagent indicated that PesO stereoselectively hydroxylates carbon 3 of Pro18. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 7 Figure 2. HR-MS/MS analysis of unmodified precursor peptide PesA2 or PesA2 co- expressed with the indicated modifying enzymes. The MS/MS fragmentation pattern of each modified peptide is shown (for tandem MS spectra, see Figure S6). Prior to analysis, the peptides were digested with endoproteinase GluC. Fragment ion annotation w as performed using the interactive peptide spectral annotator 43 with residues indicated in lower case p and d entered as hydroxylated residue s (M+16), and in lower case d n as two residues that were hydroxylated and nitrile containing (+16 and −19 to give a net change of −3 Da). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 8 Figure 3. Structural determination of PesA2 co-expressed with PesOHIC. a) LC-MS analysis of hydroxyaspartate authentic standards or hydrolyzed PesA2 -PesHI and PesA2-PesOHI peptides derivatized with L-FDAA; for coinjections, see Fig. S7. b) LC-MS analysis of L-FDAA derivatized hydroxyproline from authentic standards or hydrolyzed PesA2-PesHI and PesA2-PesOHI peptide s; c) 1H−13C HMBC spectrum of digested PesA2-PesHIC highlighting the C-terminal residue, and d) proposed structure of digested PesA2-PesHIC peptide 1 and PesA2-PesOHIC peptide 2 based on NM R, MS/MS and Marfey’s analysis. Key HMBC correlations from c) are labeled. Incorporation of the AS-like enzyme PesC into the co-expression system resulted in a new product with a −19 Da mass shift relative to the PesOHI modified peptide. HR - MS/MS analysis demonstrated that a −3 Da mass shift had occurred to the C -terminal Asp21-Asn22 sequence (Figure 2 and S6). Asparagine synthetases usually catalyze the amidation of aspartate to yield asparagine, 44 while in natural product biosynthesis, members of this enzyme family also catalyze lactam formation. 45,46 Therefore, we envisioned that an amidation reaction of a carboxyl group (−1 Da) and a dehydration .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 9 event ( −18 Da) would account for the observed overall mass shift of −19 Da . When coupled to the PesHI catalyzed hydroxylation of Asp21 (+16 Da) , amidation and dehydration would explain the net −3 Da shift of the Asp21 -Asn22 motif. Within this sequence, the dehydration could originate in a dehydroAsp, or in the formation of ester, imide, or nitrile group s ( Figure S 8). The t andem MS/MS data were inconclusive in distinguishing these possibilities, and we therefore turned to analysis by nuclear magnetic resonance (NMR) spectroscopy described in the next section. We also integrated the hypothetical protein PesX into the co-expression system. Co- expression of PesX in any combination with the other enzymes did not lead to the emergence of any new species derived from PesA2. These results imply that PesX was either non-functional in the E. coli heterologous host or the protein serves a non-catalytic role in the pes BGC. As described below, a potential function of PesX is suggested from in vitro experiments. As noted above, c o-expression experiments of PesA1 with Pes enzymes yielded similar modification patterns as PesA2 (Figure S9), indicating that the less conserved precursor N-termini as well as the residue at position 20 (Thr/Val) have minimal impact on the function of the modifying enzymes within the pes BGC. Structural Elucidation of Modified PesA2 The data described thus far suggest that PesC catalyze s consecutive amidation/dehydration reactions on the precursor peptide that is hydroxylated by PesHI on Asp21 , yet the precise form of dehydration remain ed unclear. For NMR structural determination, we first attempted to process the modified PesA2 peptides with the native protease PesP1/P2. While the expression of this heterodimer in E. coli yielded soluble protein (Figure S10), prolonged incubation of the peptides and PesP2/P1 led to a range of proteolytic products, with a 25-mer the major product (Figure S11). The poor in vitro activities of PesP2/P1 limited their application in this study, and therefore we mutated Gln14 to Lys in PesA2 with the aim of generating a short peptide upon digestion with endoproteinase LysC (for residue numbering, see Figure 1a). We first explored the impact of this mutation on the PTM process, as well as the minimal enzyme requirement for .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 10 complete C -terminal modification. HR-MS/MS analysis of this Q14K mutant after co - expression with PesHIC verified that the product still contained the −3 Da change in the Asp21-Asn22 sequence (Figure S12), indicating that Gln14 is not required for PesHIC modification. Therefore, we conducted large scale preparation of PesHI C-modified PesA2-Q14K in E. coli. After IMAC purification and LysC digestion , the resulting octapeptide was purified by high-performance liquid chromatography (HPLC) and used for NMR spectroscopic analysis. A combination of 1D and 2D NMR experiments was conducted to elucidate the structure. Based on interactions observed in 1H-1H TOCSY, 1H-1H NOESY, 1H-13C HSQC and 1H-13C HMBC spectra (Figures S13-S15), all proton and carbon signals from each of the eight residues were assigned ( Table S1). Importantly, all side chain protons and carbons of the Asp21-Asn22 sequence displayed chemical shift deviation compared to unmodified Asp/Asn residues. For Asp21, the β-carbon shifted downfield to 71.2 ppm and has only one proton attached to it, affirming that PesHI catalyze d β-hydroxylation o f Asp21. Thus, the NMR data ruled out dehydration involving the hydroxyl group introduced by PesHI, and in turn implicated that the −19 Da change must occur entirely on the C - terminal Asn. T he α-proton of Asn22 shifted downfield to 5.13 ppm, indicating a more electron-withdrawing environment. Together with the overall mass change, these findings are consistent with co nversion of the C -terminal carboxylate into a nitrile group. Consistent with this hypothesis , the 13C NMR spectrum showed only 10 rather than the expected 11 signals in the range of 150-220 ppm (10 amide/carboxylate carbonyl carbons and one guanidine carbon), and instead a characteristic nitrile carbon signal at 118.0 ppm was observed. In the 1H-13C HMBC spectrum, this carbon signal showed correlations with the α and β protons and the amide proton of former Asn22 (Figure 3c). Collectively, these observations from 1D and 2D NMR experiments unambiguously show that in the PesA2- PesHIC peptide, the C-terminal carboxylate is converted to a nitrile group. We also isolated and characterized the precursor peptide PesA2-Q14K after PesOHIC modification and LysC digestion by NMR spectroscopy (Figure S1 6-S18). Compared with the PesA2 -PesHIC peptide, the signals characteristic of a β- hydroxyaspartate as well as the C-terminal nitrile group were retained, and additional chemical shift deviations from unmodified residues were mainly observed for protons and .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 11 carbons associated with the Pro residue (Table S2). In the 1H-1H COSY and 1H-13C HSQC spectra, the observed correlations clearly assigned the hydroxy group to the β position (C3), consistent with the conclusions from Marfey’s analysis . This modification resulted in the conversion of a CH2 group to a CH group, accompanied by a downfield shift of the β-proton to 4.43 ppm and the corresponding β-carbon to 73.2 ppm. Characterization of the Nitrile Synthetase PesC With the C-terminal nitrilation activity of PesC established, we examined the reaction in vitro . For typical AS-like enzymes , the N -terminus is buried inside the enzyme .44 Therefore, we constructed a PesC-encoding plasmid with a C -terminal His 6-tag for expression in E. coli. In the canonical AS catalytic cycle, L-aspartate is converted to L- asparagine via ATP-dependent adenylation of the side chain carboxylate , followed by amidation using L-glutamine as the ammonia donor.44 Because PesC shows sequence homology to asparagine synthetases, IMAC-purified PesC was incubated with Mg2+, ATP, L-glutamine and the PesA2-PesOHI peptide for 6 hours. LC-MS analysis of the GluC - digested product clearly demonstrated the formation of the C-terminal nitrile (Figure 4a). When ATP was omitted from the reaction, nitrilation activity was abolished. When the assay was performed for 1 hour, a product with a mass shift of −1 Da was observed by high resolution electron spray ionization mass spectrometry (HR-ESI MS) (Figure 4a) consistent with the formation of a n intermediate C-terminal amide. To confirm the origin of the nitrogen atom in both products, the assay was also performed with L-glutamine- (amide-15N). The resulting C-terminal amide and nitrile containing peptides were both detected by LC-HRMS with a +1 Da mass shift (Figure 4b), indicating that in the PesC reaction, the inserted nitrogen is indeed derived from the side chain amide nitrogen of glutamine. Next, we explore d the substrate requirements of PesC. The importance of prior PesHI/PesO modification for PesC activity was first examined. When unmodified precursor PesA2 , PesA2-PesHI, and PesA2-PesOHI were incubated individually with PesC, the production of the corresponding nitrile products was only observed with PesHI- and PesOHI-modified PesA2 (Figure 4a, S19). For unmodified PesA2, only trace amounts .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 12 of the amide species were formed. Thus, the hydroxylation of the adjacent Asp residue on the substrate is essential for efficient amidation and for dehydration of the intermediate amide to occur, while hydroxylation by PesO is not required. This conclusion is consistent with the gene composition of orthologous BGCs, because pesHI homologs always co - occur with pesC homologs while pesO is only partially conserved (e.g. Figure S4). The leader peptide dependence of PesC was examined using PesA1 as substrate, which provided more PesOHI -modified product from the co -expression experiment. We purified PesOHI-modified, endoproteinase LysC digested PesA1 -Q14K as well as PesOHI-modified, endoproteinase AspN digested PesA1. Reaction of the C-terminal 14- mer (AspN product) with PesC under the standard reaction conditions resulted in nitrile production (Figure S20). The C-terminal 8-mer (LysC product) was converted by PesC to the corresponding amide with a small amount of nitrile formed (Figure S20). These results suggest that PesC can still function in the absence of a leader peptide, but the attenuation in PesC efficiency suggests that the enzyme likely acts prior to proteolysis in the biosynthetic pathway. Figure 4. a) LC-MS analysis of the in vitro PesC reaction with PesA2 co-expressed with PesOHI after GluC digestion (assay conditions: 100 μM PesA2-PesOHI peptide, 100 μM PesC, 5 mM ATP, 10 mM L-glutamine, 20 mM Mg2+); b) HR-ESI MS analysis of the resulting amide or nitrile species when L-glutamine or L-glutamine-(amide-15N) was used in the assay (calculated mass for amide product [ M+2H]2+: 1111.5798, nitrile product [M+2H]2+: 1102.5745). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 13 Based on these observations, a mechanism for the PesC catalyzed nitrilation can be proposed (Figure S21). The reaction is initiated by ATP-dependent adenylation of the C- terminal carboxylate, followed by the nucleophilic attack of glutamine-derived ammonia to form a C-terminal amide intermediate. The amide is then activated by a second equivalent of ATP to generate an AMP-imidate, from which AMP is eliminated to yield the C-terminal nitrile. This mechanism is similar to that previously proposed for AS-like nitrile synthetases in other biosynthetic contexts such as the NRPS product auranthine and 7- cyano-7-deazaguanine (preQ0).13,14,47 With the aim of potentially uncovering more AS-like enzymes that are capable of installing nitrile groups on ribosomal peptides, we performed a bioinformatic analysis on PesC. A BLASTp search with the PesC sequence as query only recovered high-identity homologs of PesC located in orthologous clusters of the pes BGC. Other less highly conserved hits were not related to RiPP biosynthesis, e.g. enzymes linked to the biosynthesis of N-acetylglutaminylglutamine amide.48 We then constructed a sequence similarity network (SSN) of the asparagine synthase protein family (PF00733) , and examined their co -occurrence with RiPP biosynthesis elements ( Figure S22).49,50 This analysis identified ~3,800 candidates, of which ~3,500 were related to lasso peptide biosynthesis. Among the remaining enzymes, TsrC51 and ScdTA52 are known to catalyze amidation of the C-terminal carboxylate of their substrate peptides. The remaining ~300 uncharacterized candidates suggest a potential underexplored functional space of AS- like enzymes in RiPP biosynthesis. These enzymes may catalyze amidation, nitrilation, lactam formation, or currently uncharacterized reactions. In vitro Characterization of MNIO Proteins The co-expression experiments were unable to assign a role for the MNIO-like protein PesX. We therefore conducted in vitro characterization. Considering the prevalent partner dependence of MNIO proteins in the literature,39 we constructed PesHI and PesXI expression plasmid s with a His6-tag on the N -terminus of either PesH or PesX. As anticipated, PesI was co-purified with both PesH and PesX (Figure 5b). Reaction of PesA2 with as -purified PesHI resulted in hydroxylation similar to the co -expression .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 14 studies. When the assay was performed with PesH (no PesI) or with PesXI, no activity was observed by ESI-MS (Figure 5a). These findings are consistent with the absence of the iron-binding amino acids that are characteristic for MNIOs in the sequence of PesX (Figure S3), but do not provide any insights regarding the potential function of the protein. The ability of PesX to co-purify with partner protein PesI led us to speculate that PesX might serve to regulate PesH activity, and thereby nitrile formation . Structural models of the PesXI and PesHI heterodimer s using AlphaFold 3, and alignment of these two structures revealed strong similarity (Figure 5c). Similar to reported structures of the TglHI heterodimer,53 only the helical N-terminus of PesI interacts with PesH or PesX . The overlapping binding interface suggests that PesX can compete with PesH in binding to the partner protein PesI, which is critical for PesH activity as shown above. This proposal is supported by the observation that introduction of PesX in the PesHI activity assay resulted in diminished hydroxylation activit y (Figure 5a) . Moreover, addition of Strep- tagged PesX to a solution of the His6-PesH/PesI heterodimer, and subsequent addition to Ni-NTA resin resulted in the complex of PesX and PesI eluting from the column (Figure 5b). These observations indicate that PesX compete s with PesH in binding with partner protein PesI, thus inhibiting its aspartate hydroxylation activity. Considering Asp2 1 hydroxylation is essential for PesC to perform nitrile formation, expression of PesX can slow down the production of the nitrile product. If correct, this regulatory mechanism suggests that the nitrile product may be toxic to t he producing organism and that its production must be delicately regulated. We performed bioinformatic analysis on PesX to gauge its distribution . A BLASTp search with an E value cutoff of 0.05 retrieved only PesX homologs from orthologous pes- like BGCs, suggesting that the sequence of PesX is highly specific to the nitrile formation pathway. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 15 Figure 5. PesX inhibits the activity of PesH. a) ESI-MS analysis of the in vitro PesH/X/I reaction with PesA2 peptide (calculated mass for unmodified product [ M+7H]7+: 1002.6476, hydroxylated product [ M+7H]7+: 110 4.9326); b) SDS-PAGE analysis of purified His 6-PesH/PesI, His 6-PesX/PesI, and Strep-PesX. Also shown are the flowthrough (FT) and elution (E) fractions of His6-PesH-PesI and Strep-PesX loaded on an IMAC column; c) Overlayed AlphaFold 3 models of the PesHI and PesXI complexes. Nitrilobacillin Inhibits Cysteine Proteases While nitrilobacillin represent s the first peptide natural product with a C -terminal nitrile group, such scaffolds have been extensively explored in medicinal chemistry. As seen in the therapeutic peptides vildagliptin and nirmatrelvir (Figure 6),6,7 nitrile groups are common warheads in peptide -derived compounds that function by covalently inhibiting cysteine/serine proteases. To assess such activity of pes BGC products, peptide 2 (Figure 3d), was evaluated with a panel of proteases. Compound 2 exhibited little to no inhibitory activity against trypsin, chymotrypsin or angiotensin-converting enzyme (ACE) at 25 μM concentration. Instead, it inhibited the cysteine proteases cathepsin L, cathepsin B and papain with IC 50 values of 1.1 μM, 9.0 μM and 9.7 μM, respectively (Figure 7) . These observed inhibitory activities align well with the reported activities of synthe tic peptidyl nitriles ,7 and highlight the poten tial of engineering nitrilobacillins as cysteine protease inhibitors. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 16 Figure 6. Proposed biosynthetic pathway of nitrilobacillin . Representative examples of therapeutic peptides with C-terminal nitrile groups are shown in the box. Figure 7. Protease inhibition assay of compound 2 with human cathepsin L (top), human cathepsin B (middle) and papain (bottom). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 17

In genome mining campaigns for RiPP BGCs, a common strategy features a RiPP class-defining enzyme family as query.23 This approach has proven an efficient means to discover new and divergent compounds in existing RiPP families. Several ubiquitous families of RiPP post -translational modification enzymes are not currently identified as class-defining because they occur in many different RiPP classes. Two such enzyme families are the MNIO and αKG-HExxH enzymes.28,39 These proteins catalyze a variety of different transformations that at present cannot be predicted. In the current study, we investigated a representative BGC featuring members of both protein families that lead to the identification of the nitrilobacillins (Figure 6). Since the nitrile functional group is new to RiPPs and most likely functions as the pharmacophore of the mature products, we suggest the name nitrilotides for the wider group of products and the AS-like enzymes as the class-defining enzyme. In medicinal chemistry, nitrile groups are common ly used as warheads including in in peptide-derived drug candidates. For instance, the nitrile s effect covalent inhibition of cysteine/serine proteases in vildagliptin and nirmatrelvir (Figure 6).6,7 Similarly, the C - terminal nitrile containing peptide product nitrilobacillin (compound 2) characterized in this work exhibited micromolar inhibition activities against several cysteine proteases. These observations highlight the convergence between synthetic , designed inhibitors and biosynthetic evolution. Currently, only moderate inhibitory activity was observed with peptide 2, likely because the investigated proteases are not its physiological targets. Typically, peptidic protease inhibitors have structures at the P1-P3 position that resemble the native substrates. For instance, the pyrrolidone moiety at the P1 position of nirmatrelvir mimics the glutamine residue recognized by SARS -CoV-2 main protease. 16 These observations suggest that the currently unknown physiological targets of nitrilobacillins are proteases that cleave at asparagine residues. αKG-HExxH proteins were recently identified to be iron and α -ketoglutarate dependent dioxygenases. 28 Unlike canonical Fe /αKG dependent enzymes, proteins within this family utilize a conserved HExxH motif to bind iron , yielding a new active site architecture.28,32 The reactivity of PesO described in the current work is consistent with the reported β-hydroxylation activities of previous studies on members of the αKG-HExxH .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 18 enzyme family,28,32 while revealing proline as a new site of modification since previous members hydroxylated His, Asp, Phe and Gln residues. Hydroxylations of proline at the β/γ carbons are common modifications in the contest of single amino acid and protein side chain s, and are usually catalyzed by canonical Fe/ αKG enzymes. 54 In RiPP biosynthesis, proline β,γ-dihydroxylation has been reported in the biosynthesis of microbisporicin and is catalyzed by the cytochrome P450 enzyme MibO.55 The AS-like enzyme PesC catalyzes the nitrilation at the C-terminal carboxylate in the PesA substrates expanding the previously reported small number of ATP-dependent nitrilation enzymes, ToyM/QueC47 and ArtA/NitB.13,14 These enzymes introduce nitriles into nucleoside bases and the side chains of amino acids. Other pathways to nitrile containing natural products involve oxidation of amino groups by hemeproteins9,10 and FMOs,12 as well as oxidative rearrangement processes of -amino acids by non -heme iron dependent enzymes (Figure S1).56 Given the structures of their substrates, from a biocatalyst development perspective, PesC may present the most promising starting point for enzymatic installation of nitriles at the C-terminus of peptides. The reaction catalyzed by PesC has some differences compared to the two previous ATP-dependent systems. QueC/ToyM utilizes ammonia as the nitrogen source instead of Gln used by P esC, whereas ArtA/NitB catalyzes only the amide-to-nitrile dehydration step. Crystallographic structures of QueC and ArtA have been solved,13,57 and alignment of the AlphaFold 3 predicted structures of PesC with the crystal structures of QueC or ArtA reveals that, even though chemically the transformation catalyzed by PesC and QueC are more similar, the overall fold of PesC is better aligned with ArtA (Figure S23). More specifically, the ATP binding domain of PesC exhibit s structural similarity with th at of ArtA. However, the substrate binding pocket of PesC is different from the active site of ArtA, and the key amide-interacting residues (Q146, S197, D199) identified through docking and mutational analysis of ArtA13 are not conserved in PesC. Instead, the active site of PesC adopts a much more open conformation in the model , likely to allow accommodation of large peptide substrates (Figure S23). Enzymes from the AS protein family catalyze key transformations in the biosynthesis of bioactive natural products, such as β-lactamization in the biosynthesis of several β- lactam antibiotics.45,58,59 In RiPP biosynthesis, the lasso peptide cyclases belong to th e .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 19 AS enzyme family .46,60 In addition, several characterized asparagine synthetase -like enzymes such as TsrC51 and ScdTA52 amidate the C-termini of their substrate peptides. The nitrilation reactivity of PesC identified herein expand s the diversity of chemical outcomes of asparagine synthetase-like enzymes in RiPP biosynthesis . Our current genome mining efforts identified hits that are likely involved in novel RiPP biosynthesis, but no homologs were predicted with high confidence to catalyze nitrile formation, in part because it is currently not clear what controls enzymatic one-step amidation versus two- step nitrilation. Multinuclear non-heme iron dependent oxidative enzymes (MNIOs) are an emerging class of metalloenzymes that contain two or three iron ions in their active site.39 Although this protein family comprises more than 14,000 members, only a handful have been functionally characterized. MNIOs are strongly associated with the tailoring of RiPPs. They catalyze a wide array of novel oxidative modifications to construct unusual scaffolds including oxazolones and thioamides,33 thiooxazoles,35,36,61 and ortho-tyrosines.30 PesHI is shown here to catalyze a comparatively simpler transformation, the stereoselective - hydroxylation of aspartate to 3 S-hydroxyAsp. This same reaction was also recently reported for an MNIO -nitroreductase fusion enzyme PflD where the MNIO domain catalyzes the transformation.32 In another related system, ApyHI catalyzes the oxidation of a C-terminal Asp residue to the corresponding alpha-keto acid with a β-hydroxylated aspartate as a proposed intermediate.27 Sequence alignment of Pes H, ApyH and PflD revealed less than 30 % identity for the MNIO domain . Notably, the conserved binding HxD motif that normally binds the third iron (Fe3) in MNIOs is missing for both PesH and PflD (Figure S3). Previous mutational studies on the MNIOs MbnB62 and TglH53 showed that mutating some Fe3 binding residues lead to decreased but not abolished enzymatic activity. Thus, the role of Fe3 in characterized MNIO enzymes remains elusive. The function of the MNIO-like protein PesX is intriguing. No activity was detected in vitro or in E. coli, consistent with the absence of the metal binding residues in its sequence that are conserved in all other MNIOs. Heterodimeric proteins composed of an inactive homolog with an active enzyme is not uncommon and is observed for instance with the Fe/αKG enzyme system CorB-CorD,31 and with several examples of metalloproteases.63- 66 However, PesX appears to function in a different manner in that it competes with PesH .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 20 for PesI, which is required for PesH activity . The identification of separate operon s governing pesH and pesXI expression, whereas in other MNIO systems the HI proteins are often under control of a single operon (Figure S24), suggests that PesH activity is regulated in a more complicated manner. PesC catalyzed nitrilation only after hydroxylation o f Asp21 by PesHI, suggesting that this hydroxylation might serve as a gatekeeping event. The observed competition of PesX fo r binding PesI could therefore be relevant to tightly control nitrile formation within the native host, possibly to avoid toxicity. In this model, PesX and PesI would be expressed resulting in a non -functional heterodimer, which then would be converted to a fraction of ac tive PesHI heterodimer upon initiation of PesH expression. Experimental test of this suppression strategy in species harboring pes-like BGCs requires investigations with native producing strains.

In this work, we identified and characterized a novel RiPP BGC from Peribacillus simplex. The activity of modifying enzymes w as assigned through a combination of HR -ESI- MS/MS, NMR and Marfey’s analysis . MNIO and αKG-HExxH proteins were shown to perform stereoselective β-hydroxylation on aspartate and proline residues, respectively , and the AS-like enzyme PesC was demonstrated to catalyze two-step nitrile formation at the C-terminus of ribosomally produced peptides. The resulting peptide exhibits inhibitory activity against several cysteine proteases. This study therefore expands the list of ribosomal peptide PTMs with nitrilations , and unveils a convergence between rational molecule design and natural evolution. Supporting Information Experimental procedures, Figures S 1-S24 showing spectroscopic data , AlphaFold 3 modeling, and Tables S 1-S3 listing NMR annotations and protease inhibition assay conditions. Funding .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 21 This manuscript is the result of funding in whole or in part by the National Institutes of Health (NIH ; grant R37 GM058822 ). It is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH . A Bruker UltrafleXtreme mass spectrometer used was purchased with support from the Roy J. Carver Charitable Trust (Grant No. 22 -5622). W.A.v.d.D. is an Investigator of the Howard Hughes Medical Institute. Notes The authors declare no competing financial interest.

The authors thank Dr. Dinh T. Nguyen for helpful discussions, and Prof. Angad P . Mehta for access to an Agilent Synergy H1 plate reader. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 14, 2026. ; https://doi.org/10.64898/2026.03.11.711119doi: bioRxiv preprint 22

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