Backstory: During my postdoc under "Brandon I. Morinaka" at the National University of Singapore (2021-2023), I was deeply inspired and influenced by his dedication in searching for new antibiotics, xenorceptides targeting BamA, an essential outer membrane protein of Gram-negative bacteria.[1,2] Xenorceptide has a structural characteristic of cyclophane rings, which is formed by cross-linking between an aromatic side chain and one aliphatic amino acid at the three-residue motif. The cyclophane rings on the peptides are installed by radical SAM enzymes, which has an extended C-terminal region containing a second domain called SPASM. In the biosynthesis, xenorceptides are produced following defined RiPP biosynthetic rules, (1) the xenorceptide backbone peptide is encoded at the C-terminal of a precursor peptide, (2) the radical SAM enzyme found in the flanking regions of a precursor peptide install cyclophane rings on the xenorceptide backbone peptide, and (3) the protease found in the gene cluster cleave the N-terminal peptide region and release the cyclophane-containing xenorceptide backbone as a mature bioactive natural product.[3] However, in many cases, these radical SAM-RiPP biosynthetic gene clusters (BGCs) do not contain proteases, hindering the exploration of the mature natural products and their associated bioactivities.[4]
Discovery of S9 protease WprP2 : Inspired by my former lab, in this study, we aims to identify putative natural products from cyclophane-containing precursor peptides generated by the radical SAM enzyme WprB1.[5] To do that, we first searched for the presence of a protease in the flanking region of WprB-homologs. Unexpectedly, we identified an uncharacterized serine protease WprP2 from Streptomyces venezuelae NPDC049867, encoded next to the radical SAM enzyme WprB2 involved in the cross-linking between Trp-C5 and Arg-Cγ at the WPR motif. WprP2 catalyzes the uniform cleavage on the precursor peptide WprA2, with the first cleavage occurring after WPR motif and second cleavage occurring before the Pro amino acid at the 12th residue preceding the WPR motif. Such cleavage has not been seen in any serine proteases from RiPP biosynthesis. In addition, we also performed the following studies: (1) site-directed mutagenesis to identify the recognition sequence and catalytic residues of enzyme, (2) substrate scope to determine the enzyme's tolerance to substrate composition and length, and (3) computational modeling to predict the first and second cleavage sites. This study has now reported in Communications Chemistry.[6]
Moving forward, the discovery of the S9 protease WprP2 will enable us to pursuit for new antibiotics from the end products of this pathway, while the short-term goal is to characterize other post-translational modification enzymes found in the BGC.
References:
1. Modaresi S. M.; Sugiyama R.; Tram N. D. T.; Jakob R. P.; Phan C.-S.; Saei A. A.; Morishita Y.; Mühlethaler T.; Lim J.; Ritz D.; Long P. S. Y.; Lehner P. A.; Lim Z. H.; Degen M.; Yao Z.; Maier T.; Hou Y.; Lee J. Y.; Xu J.; Yeat A. Y. J.; Koh K. T. S.; Goh W. Y.; Ling S. Y. H.; Chua P. W. L.; Yamazaki M.; Ee P. L. R.; Hiller S.; Morinaka B. I. Antibiotics that kill Gram-negative bacteria by restructuring the outer membrane protein BamA. bioRxiv 2024. https://doi.org/10.1101/2024.12.16.628070
2. Morinaka B. I.; Sugiyama R.; Yao Z.; Ee P. L. R.; Tram D. T. N.; Morishita Y.; Phan C.-S.; Lim J. Peptides with antimicrobial properties. 2024, PCT patent no. PCT/SG2023/050524. https://patentscope.wipo.int/search/en/WO2024025474
3. Nguyen, T. Q. N.; Tooh, Y. W.; Sugiyama, R.; Nguyen, T. P. D.; Purushothaman, M.; Leow, L. C.; Hanif, K.; Yong, R. H. S.; Agatha, I.; Winnerdy, F. R.; Gugger, M.; Phan, A. T.; Morinaka, B. I. Post-translational formation of strained cyclophanes in bacteria. Nat. Chem. 2020, 12, 1042–1053. https://doi.org/10.1038/s41557-020-0519-z
4. Phan C.-S.; Morinaka B. I. Bacterial cyclophane-containing RiPPs from radical SAM enzymes. Nat. Prod. Rep. 2024, 41, 708-720. https://doi.org/10.1039/D3NP00030C
5. Khan A. H.; Haedar J. R.; Kiselov V.; Romanuks V.; Smits G.; Donadio S.; Phan C.-S. Radical SAM enzyme WprB catalyzes uniform cross-link topology between Trp-C5 and Arg-Cγ on the precursor peptide. ACS Chem. Biol. 2025, 20, 259-265. https://doi.org/10.1021/acschembio.4c00693
6. Haedar J. R.; Khan A. H.; Ma S.; Donadio S.; Phan C.-S. Protease WprP2 catalyzes uniform cleavage on the precursor peptide in RiPP biosynthesis. Commun. Chem. 2026. https://doi.org/10.1038/s42004-026-01915-w