Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM

Feb 13, 2017 - We report the characterization of both radical SAM enzymes and find that they contain multiple [4Fe-4S] clusters and catalyze Lys-Trp c...
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Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci Kelsey R Schramma, and Mohammad R. Seyedsayamdost ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01069 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci

Kelsey R. Schramma† and Mohammad R. Seyedsayamdost†,‡,* Departments of Chemistry† and Molecular Biology‡, Princeton University, Princeton, NJ 08544

*email: [email protected]



 

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Abstract Macrocycles represent a common structural framework in many naturally-occurring peptides. Several strategies exist for macrocyclization, and the corresponding enzymes are of great interest, as they enhance our repertoire for creating complex molecules. We recently discovered a new peptide cyclization reaction involving a crosslink between the side-chains of lysine and tryptophan that is installed by a radical SAM enzyme. Herein, we characterize relatives of this metalloenzyme from the pathogens Streptococcus agalactiae and Streptococcus suis. Our results show that the corresponding enzymes, which we call AgaB and SuiB, contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective substrates. Subsequent high-resolution-MS and 2D-NMR analyses located the site of macrocyclization. Moreover, we report that AgaB can accept modified substrates containing natural or unnatural amino acids. Aside from providing insights into the mechanism of this unusual modification, the substrate promiscuity of AgaB may be exploited to create diverse macrocyclic peptides.



 

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Text Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a growing class of secondary metabolites with a wide range of bioactivities and diverse, often cyclic, structures.1,2 Investigations of the biosynthesis of RiPPs have revealed numerous new transformations, and these metabolites thus represent sources of novel small molecules and enzymatic chemistries.1-4 One such example is streptide, which is produced from the str gene cluster by Streptococcus thermophilus (Fig. 1A).5,6 The str cluster encodes a precursor peptide, StrA, a radical S-adenosyl-methionine (SAM) enzyme, StrB, and a protease, StrC. We recently reported the structure and biosynthesis of streptide, the founding member of a new class of RiPPs that contain an unusual Lys-Trp crosslink (Fig. 1).5 We found that the crosslink is installed by StrB at the β-carbon of Lys and the side-chain-C7 of a Trp residue in the precursor peptide, StrA. Proteolytic cleavage at the N- and C-termini of crosslinked StrA, catalyzed by StrC (possibly in conjunction with a second protease), furnishes the mature macrocyclic 9mer product. By searching the existing microbial genome database, our investigations further showed that a number of human pathogens also harbor str-like gene clusters, suggesting that they too produce Lys-Trp crosslinked peptides.5 Herein, we assess this hypothesis using a biochemical approach, in which we examine StrB homologs from two human pathogens, Streptococcus agalactiae and Streptococcus suis. We report the characterization of both radical SAM enzymes and find that they contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective precursor peptides in a leader peptide-dependent fashion. Examinations of the substrate specificity in one case provide additional insights into this unusual post-translational modification. Bioinformatic searches show that three pathogenic Streptococci, S. agalactiae, S. suis, and Streptococcus mitis, contain a str-like gene cluster, which we have named aga, sui, and mit, respectively (Fig. 1B). In all cases, the gene cluster is preceded by a shp-rgg quorum sensing (QS) module,6-8 which suggests that the corresponding mature product is generated as a function of QS at high cell densities, although none of these QS systems has been examined experimentally. S. agalactiae is an opportunistic human pathogen, especially prevalent in neonatal infections.9 S. suis is primarily a pig pathogen and a zoonotic agent capable of transferring to humans.10 S. mitis is the causative agent of endocarditis and can lead to fatal infections.11 We focused our efforts on the radical SAM enzymes AgaB and SuiB from S.



 

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agalactiae and S. suis, respectively (Fig. 1B). These enzymes were cloned, expressed, purified, and subsequently characterized anaerobically. Radical SAM enzymes typically contain a [4Fe-4S] cluster at the active site, which is held in place by an N-terminal CxxxCxxC motif.12-14 Reaction of the reduced form of this cluster with SAM results in formation of a 5ꞌ-deoxyadenosyl radical (5ꞌ-dA•), which initiates catalysis by abstraction of a hydrogen atom from the substrate. StrB, AgaB and SuiB belong to the SPASM class of radical SAM enzymes, named after their founding members (subtilosin, PQQ, anaerobic sulfatase, and mycofactocin).15,16 These enzymes contain up to two auxiliary Fe-S clusters, referred to as AuxI and AuxII, which are bound by Cys-rich C-terminal motifs (Fig. 1B).17-23 We previously reported that StrB contains two [4Fe-4S] clusters, one N-terminal and one C-terminal bound by the CxxxCxxC and SPASM motifs, respectively. We identified two Cys residues in the SPASM domain, C409 and C415, required for ligating an auxiliary cluster. An alignment of the sequences of StrB, AgaB, and SuiB suggests that these new homologs would also bind at least two [4Fe-4S] clusters (Fig. 1B). To determine the number and location of Fe-S clusters in AgaB and SuiB, we employed a strategy in which two Cys residues in each predicted Fe-S binding site were substituted by Ala to abrogate cluster binding at that position. The mutants were then expressed, purified, reconstituted, and characterized by quantitation of Fe and labile sulfide, as well as by UV-vis and EPR spectroscopies. The following mutants were generated: C120A/C123A-AgaB and C121A/C124A-SuiB, to remove the N-terminal Fe-S cluster bound by the CxxxCxxC motif, as well as C408A/C414A-AgaB and C409A/C415A-SuiB to remove binding of a putative AuxII cluster (Table S1). All four mutants were expressed and purified successfully. We previously attempted to remove the AuxI site in StrB using a C347A/C365A-StrB variant, but were unable to obtain soluble protein. Because of the high sequence similarity to StrB (>90%), we did not attempt to generate the corresponding AuxI mutants in AgaB and SuiB. UV-vis absorption spectra of purified wt AgaB and SuiB both exhibit bands at 280 nm, associated with the protein, as well as a shoulder at 320 nm and a broad peak at 395 nm, which are characteristic for an oxidized [4Fe-4S]2+ cluster (Fig. 2A, B). Quantitative analysis of iron and labile sulfide of the as-isolated wt AgaB and SuiB enzymes yielded 6.6 ± 0.1 Fe and 5.9 ± 0.3 S (AgaB) and 6.6 ± 0.1 Fe and 6.2 ± 0.2 S (SuiB). Reconstitution of the proteins with 20-fold excess iron and sulfide resulted in 9.0 ± 0.3 Fe and 8.5 ± 0.6 S (AgaB) and 10.4 ± 0.1 Fe and 9.0 ± 0.1 S (SuiB, Figs. S1-S2, Table S2). These data indicate that both AgaB and SuiB contain at least two Fe-S clusters. 4 

 

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UV-vis spectra of the Cys-to-Ala double mutants displayed lower absorption at 320 nm and 395 nm relative to 280 nm (compared to wt), consistent with a decrease in the Fe-S cluster content (Fig. 2A, B). Further, measurement of Fe and labile S for C120A/C123A-AgaB gave 4.1 ± 0.3 Fe and 4.5 ± 0.1 S per protein, while C121A/C124A-SuiB gave significantly higher Fe (7.2 ± 0.1) and labile S (6.7 ± 0.2 S) concentrations (Table S2). These results seem to point to three total [4Fe-4S] clusters, especially in the case of SuiB. Further evidence for this proposition came from the auxiliary cluster mutants, where reconstituted C408A/C414A-AgaB contained 4.3 ± 0.2 Fe and 5.3 ± 0.2 S per protein and C409A/C415A-SuiB gave 6.7 ± 0.1 Fe and 6.0 ± 0.1 S per protomer. We further examined the reconstituted wt and mutant proteins by EPR spectroscopy (Fig. 2C, D). Spin quantitation showed 0.26 (AgaB) and 0.25 (SuiB) equivalents of reduced [4Fe-4S]+1 per protomer. The EPR spectra of the reduced forms of these enzymes consist of a rhombic signal with gx, gy, gz of 1.89, 1.94, and 2.06 (AgaB) and 1.87, 1.94, and 2.06, (SuiB), respectively. In both cases, removal of the N-terminal [4Fe-4S] cluster, which is responsible for reductive cleavage of SAM, does not lead to significant changes in the EPR spectrum. Removal of an auxiliary cluster, however, results in the commonly observed axial signal in radical SAM enzymes, with g and g‖ of 1.94 and 2.06 for both C408A/C414A-AgaB and C409A/C415ASuiB. These results indicate that the upfield feature in the wt spectrum arises from the AuxII FeS cluster, while the other two clusters either have overlapping signals, or that the AuxI Fe-S cluster is not easily reduced in our reduction reaction. The EPR data are consistent with two or three [4Fe-4S] clusters. The Fe and labile S contents determined above favor a model in which AgaB and SuiB contain three [4Fe-4S], but additional studies by Mössbauer spectroscopy and/or X-ray crystallography are required to shed further light on this issue.18-20 Having characterized the [4Fe-4S] clusters of AgaB and SuiB, we turned our attention to their enzymatic activities. To do so, we prepared the respective substrates, AgaA and SuiA, by solid-phase peptide synthesis (Figs. S3-S4). Next, each enzyme was incubated with its substrate, SAM, and reductant, and the contents of the reaction mixture were analyzed by HPLC-Qtof-MS after a suitable reaction time. In both cases, we observed a new peak with a high-resolution (HR) mass that corresponded to a loss of 2 protons (Fig. 2E, F, and Table S3). Formation of the putative product peaks was dependent on the presence of AgaB or SuiB, SAM, and substrate peptide. The putative product accumulated in a time-dependent fashion, consistent with its assignment. We determined a Vmax/[E]T of 0.12 min-1 and 0.18 min-1 for AgaB 5 

 

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and SuiB, respectively, similar to previous results with StrB. Interestingly, we observed multiple turnovers in the absence of reductant, indicating that reducing equivalents can be recycled to catalyze multiple post-translational modifications. In support of this conclusion were 5'-dA quantification experiments in the absence of reductant, which gave 3.8 and 1.6 equivalents of the 5'-dA product relative to AgaB and SuiB, respectively. Further, with both enzymes, the leader peptide was required for product formation, as AgaA and SuiA substrate variants that lacked the leader peptide failed to yield product (Fig. 1B, Fig. S5).1,24 Collectively, these preliminary studies show that both AgaB and SuiB are active in vitro and catalyze a leaderpeptide dependent post-translational modification on their respective substrates. To elucidate the structures of the reaction products, we first carried out tandem HR-MS. We were able to observe all b and y fragment ions for the products of both AgaB and SuiB, except for those residues that are within the –KGDGW– motif, consistent with a macrocycle between the Lys and Trp residues within this sequence in AgaA and SuiA (Fig. 2G, H). In contrast, all b and y ions, including those in the –KGDGW– motif, were observed for the control reaction, in which enzyme was omitted (Figs. S6-S7, Table S4-S7). To fully elucidate the structures of the macrocyclic peptides, each reaction was carried out on a large scale. The product was isolated and subjected to 1D/2D NMR analysis (Figs. S8-S13). 1H NMR spectra in both cases were consistent with crosslink formation at the indole side chain. TOCSY, HMBC, and a selective 1D-NOESY spectrum irradiating at indole-1H6 clearly demonstrated crosslink formation at the β-carbon of the Lys residue and the C7-carbon of the indole side-chain of Trp within the –KGDGW– motif, consistent with the tandem HR-MS data (Figs. S9, S12, and Fig. 2G, H). Together, these results establish that AgaB and SuiB catalyze Lys-Trp crosslink formation in their respective substrates and extend the occurrence of this unusual posttranslational modification to peptides produced by pathogenic Streptococci. Sequence comparison of the peptide substrates StrA, AgaA, and SuiA shows that the – KGDGW– motif, where the crosslink occurs, is conserved (Fig. 1). To probe the importance of this conserved motif, we synthesized six analogs of AgaA incorporating a number of amino acid substitutions (Table S3 & Fig. 3). Two analogs, K2Nle- (norleucine) and K2A-AgaA were generated to probe the requirement of the Lys residue that undergoes the crosslinking reaction, including the terminal amino group of the Lys side-chain. The D4A-AgaA mutant was synthesized to interrogate the role of the conserved –GDG– backbone of the macrocycle. The W6Bzt- (3-benzothienyl-Ala) and W6F-AgaA mutants were created to probe the requirements 6 

 

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for the indole side-chain that is involved in the modification reaction. Finally, we tested crossreactivity of AgaB with SuiA as well as SuiB with AgaA. Interestingly, reactions with K2Nle-AgaA and K2A-AgaA failed to generate any product indicating the essential role of not just the Lys side-chain but also the Lys amino group in the reaction. On the other hand, the D4A-AgaA substrate reacted with AgaB to generate product, albeit at a ~3–fold reduced rate. While the W6F-AgaA mutant failed to give product, the W6Bzt-AgaA variant underwent crosslink formation at a rate ~4–fold lower than that of wt AgaA (Fig. 3). Finally, both AgaB and SuiB reacted with SuiA and AgaA, respectively, further underlining the substrate promiscuities observed with the AgaA variants. These results show that small C-terminal extensions can be tolerated in AgaB and have implications for the structure of its active site pocket, where crosslink formation is catalyzed. We next set out to verify the site of crosslink formation in the D4A and W6Bzt mutants of AgaA. Tandem HR-MS data were consistent with the crosslink occurring within the –KGAGW– and –KGDG(Bzt)– motifs in the two substrates (Figs. S14-S15 & Tables S8-S11). Isolation of the products and analysis by tandem HR-MS and 1D/2D NMR showed that the crosslink occurred in both cases at the β-carbon of the Lys2 residue and the C7 of the indole (in D4AAgaA) or C7 of the benzothiophene (in W6Bzt-AgaA) side-chain (Fig. 3, Figs. S16-S22). Comparison of the NMR spectra of the W6Bzt-AgaA product with commercial 7-methylbenzothiophene was consistent with this conclusion (Fig. S22). These results show that AgaB, and by extension possibly its homologs, can accommodate natural and unnatural amino acid changes in their substrates. The promiscuity in substrate recognition can be exploited in the future to generate unnatural streptide analogs in vitro and in vivo. The essential requirement of the amino group of the Lys side-chain suggests an important role during catalysis. Two scenarios may be envisioned (Scheme 1). The amino group could serve to properly position the substrate in the active site, perhaps via a salt bridge and/or H-bonding interactions (Scheme 1, pathway a). Alternatively, and more interestingly, it could be directly involved in the reaction as a Bronsted base (Scheme 1, pathway b). Our proposed mechanism involves formation of an indolyl radical intermediate, which upon deprotonation and oxidation would form product. Given its vicinity to the indolyl-C7-1H, the amino group of Lys2 in AgaA could function as a Bronsted base by removing the 1H from the indole side-chain. This process may be facilitated by an active site base. Assessment of AgaA with a variety of Lys analogs with perturbed pKas will test this model. 7 

 

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The creation of C-C bonds at unactivated carbon centers is a key challenge in chemistry and biology. We previously showed that a radical SAM enzyme, StrB, has evolved to catalyze this type of a reaction by intramolecularly stitching together the side chains of Lys and Trp during the production of streptide. Herein we demonstrate that Lys-Trp crosslink formation is not unique, and that radical SAM enzymes produced by bacteria in the human microbiome also carry out this unusual post-translational modification, suggesting that they too produce streptide variants. The responsible catalysts, StrB, AgaB and SuiB, belong to the SPASM subclass of radical SAM enzymes, an emerging enzyme family with multiple [4Fe-4S] clusters that likely participate in electron transfer reactions during catalysis. Studies addressing the promiscuity of AgaB show it to be flexible and reveal that modifications in the precursor peptide can be tolerated. These results will pave the way for generating unnatural streptide analogs with unusual C-C linkages. Such probes will not only serve to increase the chemical diversity of semi-synthetic peptides, but will also help interrogate the physiological roles of the streptidefamily of molecules, which are likely produced at high cell densities – a state associated with virulence – by pathogenic Streptococci. While further studies are required to understand the physiological roles of streptides, parallels may be drawn with the thiolactone-cyclized RiPPs of Staphylococcus aureus, AIP-II.25 Streptides may, like AIP-II variants, fulfill roles as self-strain activators and cross-strain inhibitors of QS-dependent pathways in Streptococci. Investigations of these aspects of streptide biology are underway.



 

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Methods A description of general procedures, methods used to clone wt and mutant agaB and suiB, determine protein concentrations and carry out reconstitution reactions, synthesize wt and mutant AgaA and SuiA, conduct enzymatic activity assays, and perform HR-MS, HR-MS/MS, and 1D/2D NMR on the resulting products is given in the SI. Expression and purification of AgaB and SuiB. Expression and purification of AgaB, SuiB, and the various double mutants was carried out as previously described in detail for StrB with minor modifications.5,26 The only departure from the previous procedure was the composition of the G25 buffer used to remove excess imidazole after the Talon metal affinity column. This buffer consisted of 100 mM HEPES, 300 mM KCl, 10% glycerol, 5 mM DTT, pH 7.5. Purifications of AgaB and SuiB were carried out from 17 g and 13.3 g of cell pellets respectively, and yielded 125 mg AgaB and 154 mg SuiB. A similar yield was obtained for the AgaB and SuiB double mutants. Activity Assays for AgaB and SuiB. Activity assays were generally performed in a 50 μL reaction volume and contained G25-buffer, AgaB or SuiB (25 μM), sodium dithionite (2 mM), and substrate (1 mM). At t=0, 25 μL were removed and reacted with 25 μL 100 mM H2SO4 to provide the no-reaction control. For the remaining 25 μL, the reaction was initiated by addition of 1 mM SAM, and allowed to proceed for 1 hour before quenching with 25 μL of 100 mM H2SO4. The precipitated protein was removed by centrifugation at 14,000 x g for 3 min. The supernatant was applied to a Phenomenex Strata C8 SPE column (50 mg), which had been equilibrated with H2O + 0.1% FA. The column was washed with 2 mL H2O (+ 0.1% FA), and peptides eluted with 1.5 mL of 50% MeCN in H2O (+ 0.1% FA) and 0.5 mL 100% MeCN (+ 0.1% FA). The eluates were combined, dried in vacuo, and analyzed by HPLC-Qtof-MS as described above. Percent conversions for all AgaA analogs are relative to the AgaA/AgaB reaction. In the case of SuiA/AgaB, to avoid complications arising from disparate substrate Km values, the percent conversion was determined by dividing the product/substrate ratio in the SuiA/AgaB reaction by the same ratio in the AgaA/AgaB reaction. A similar calculation was carried out for the AgaA/SuiB reaction to determine percent conversions.



 

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Associated Content Materials and Methods; Synthesis and purification of SuiA, AgaA and its analogs, HR-MS data for AgaA, AgaA analogs, and SuiA; HR-MS, tandem HR/MS, and NMR data for the products of AgaB and SuiB. This material is available free of charge via the Internet at http://pubs.acs.org.   Author Information Corresponding Author *Email: [email protected] Author Contributions KRS and MRS designed and carried out experiments, and assembled the manuscript. Notes The authors declare no competing financial interests.

Acknowledgements We thank I. Pelczer for assistance with acquisition and analysis of NMR data, and the National Institutes of Health (GM098299 to M.R.S.), the Searle Scholars Program, and the Pew Biomedical Scholars Program for support of this work. K.R.S. was supported by an Eli LilyEdward C. Taylor Fellowship in Chemistry.                      

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(10) Fittipaldi, N., Segura, M., Grenier, D., and Gottschalk, M. (2013) Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis. Future Microbiol. 7, 259-279. (11) Mitchell, J. (2011) Streptococcus mitis: walking the line between commensalism and pathogenesis. Mol. Oral Microbiol. 26, 89-98. (12) Frey, P. A., and Booker, S. J. (2001) Radical mechanisms of S-adenosylmethionine-dependent enzymes. Adv. Protein Chem. 58, 1-45. (13) Booker, S. J. (2009) Anaerobic functionalization of unactivated C-H bonds. Curr. Opin. Chem. Biol. 13, 58-73. (14) Broderick, J. B., Duffus, B. R., Duschene, K. S., and Shepard, E. M. (2014) Radical Sadenosylmethionine enzymes. Chem. Rev. 114, 4229-4317. 11 

 

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(15) Haft, D. H., and Basu, M. K. (2011) Biological systems discovery in silico: radical Sadenosylmethionine protein families and their target peptides for posttranslational modification. J. Bacteriol. 193, 2745-2755. (16) Haft, D. H. (2011) Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners. BMC Genomics 12, 21. (17) Grell, T. A., Goldman, P. J., and Drennan, C. L. (2015) SPASM and twitch domains in Sadenosylmethionine (SAM) radical enzymes. J. Biol. Chem. 290, 3964-3971. (18) Grove, T. L., Ahlum, J. H., Sharma, P., Krebs, C., and Booker, S. J. (2010) A consensus mechanism for Radical SAM-dependent dehydrogenation? BtrN contains two [4Fe-4S] clusters. Biochemistry 49, 3783-3785. (19) Goldman, P. J., Grove, T. L., Sites, L. A., McLaughlin, M. I., Booker, S. J., and Drennan, C. L. (2013) X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification. Proc. Natl. Acad. Sci. USA 110, 8519-8524. (20) Goldman, P. J., Grove, T. L., Booker, S. J., and Drennan, C. L. (2013) X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry. Proc. Natl. Acad. Sci. USA 110, 15949-15954. (21) Flühe, L., Knappe, T. A., Gattner, M. J., Schäfer, A., Burghaus, O., Linne, U., and Marahiel, M. A. (2012) the radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A. Nat. Chem. Biol. 8, 350-357. (22) Flühe, L., Burghaus, O., Wieckowski, B. M., Giessen, T. W., Linne, U., and Marahiel. M. A. (2013) Two [4Fe-4S] clusters containing radical SAM enzyme SkfB catalyze thioether bond formation during the maturation of the sporulation killing factor. J. Am. Chem. Soc. 135, 959-962. (23) Flühe, L., and Marahiel, M. A. (2013) Radical S-adenosylmethionine enzyme catalyzed thioether bond formation in sactipeptide biosynthesis. Curr. Opin. Chem. Biol. 17, 605-612. (24) Oman, T. J., and van der Donk, W. A. (2010) Follow the leader: the use of leader peptides to guide natural product biosynthesis. Nat. Chem. Biol. 6, 9-18. (25) Mayville, P., Ji, G., Beavis, R., Yang, H., Goger, M., Novick, R. P., and Muir, T. W. (1999) Structureactivity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc. Natl. Acad. Sci. USA 96, 1218-1223. (26) Lanz, N. D., and Booker, S. J. (2012) RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins. Methods Enzymol. 516, 125-152.

 

 

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Figure 1. Streptide, str gene cluster, and str homologs. (A) Structure of streptide with the LysTrp crosslink shown in blue.

(B) Comparison of the str, aga, and sui gene clusters. The

precursor peptides, radical SAM enzymes, and proteases are assigned A, B, and C, respectively.1 Also shown is a sequence alignment of the precursor peptides StrA, AgaA, and SuiA – where the core peptide is indicated in blue font – as well as comparison of the CxxxCxxC (red) and SPASM (blue) motifs in the radical SAM enzymes StrB, AgaB, and SuiB.        

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Figure 2. Characterization of wt/mutant AgaB and SuiB. (A) UV-vis spectra of wt (red), AuxII mutant (blue), and rSAM mutant (green) variants of AgaB. (B) EPR spectra of wt (red), AuxII mutant (blue), and rSAM mutant (green) variants of AgaB. Traces are color-coded as in panel A. (C) Analysis of AgaB enzymatic activity assays HPLC-Qtof-MS. The substrate (S) and product (P) peaks, as determined by HR-MS, are marked. The traces are color-coded as shown and offset in both axes for clarity. (D) UV-vis spectra of wt (red), AuxII mutant (blue), and rSAM mutant (green) variants of SuiB. (E) EPR spectra of wt (red), AuxII mutant (blue), and rSAM mutant (green) variants of SuiB. Traces are color-coded as in panel D. (F) Analysis of SuiB enzymatic activity assays HPLC-Qtof-MS. The substrate (S) and product (P) peaks, as determined by HRMS, are marked. The traces are color-coded as shown and off-set in both axes for clarity. (G, H) Analysis of the AgaB (G) and SuiB (H) products by HR-MS/MS. Observed b and y fragment are marked. No fragmentation is observed within the –KGDGW– motif (Figs. S6-S7, Tables S3-S7).

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Figure 3. Substrate analogs tested in this study. The substrate analog and enzyme used are shown in each case. All analogs were tested with AgaB, except the AgaA/SuiB reaction. Percent conversions relative to the AgaA/AgaB pair are noted parenthetically. In the case of AgaA/SuiB, the conversion is relative to the SuiA/AgaB. ‘npd’ denotes no product detected.

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Scheme 1. Possible roles for the amino group of Lys2 in AgaA during turnover.  

                       

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Table of Contents Graphic AgaA

SuiA rSAM SuiB

rSAM AgaB

 

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