Teicoplanin Nonribosomal Peptide Synthetase Is Unable to Incorporate Alpha-Ketoacid Building Blocks Minuri S. Ratnayake, Xinyun Jian, Julien Tailhades, Gregory L. Challis, Mathias H. Hansen, et al. Biochemistry, 2025 Glycopeptide antibiotics (GPAs) are a vital class of nonribosomal peptides used as therapies of last resort to treat infections by multidrug-resistant bacteria. These peptide antibiotics are assembled by nonribosomal peptide synthetases (NRPSs), modular megasynthases central to the biosynthesis of a wide range of peptide natural products. The adenylation (A) domains of NRPSs are involved in the selection and activation of the amino acid building blocks forming these peptide natural products, with their subsequent loading onto a neighboring carrier protein for incorporation into the growing peptide chain. This makes A-domains the gatekeepers of specificity in nonribosomal peptide biosynthesis, with further studies needed to reveal how this specificity is enforced at all stages of catalysis. The first building block found in GPAs is diverse and can comprise an amino acid, a ketoacid, or mixtures of both, which suggests that the A-domains responsible for selecting these residues can also incorporate non-amino acid substrates. In this study, we explored the acceptance of such substrates by the initiation module of the teicoplanin NRPS. Our in vitro assays demonstrated that this A-domain possesses an unexpected preference for activating ketoacids over the native amino acid substrate l-Hpg. However, only (d/l)-Hpg and related amino acids were able to be loaded onto the neighboring carrier protein domain during the subsequent thioesterification step. We further characterized the structure of this A-domain from teicoplanin biosynthesis in complex with d-4-hydroxyphenylglycine (d-Hpg), which revealed alterations in the positioning of the substrate carboxylate that help explain the high levels of pyrophosphate release seen with this amino acid. In combination with extensive molecular dynamics simulations, these data suggest that ketoacid incorporation in GPA biosynthesis is likely performed after amino acid incorporation by the NRPS and highlight the importance of considering both activation and carrier protein loading reactions performed by an A-domain when investigating substrate selectivity in nonribosomal peptide biosynthesis.
SRP19 and the protein secretion machinery is a targetable vulnerability in cancers with APC loss Xinqi Xi, Ling Liu, Natasha Tuano, Julien Tailhades, Dmitri Mouradov, et al. Proceedings of the National Academy of Sciences of the United States of America, 2025 Loss of the tumor suppressor gene (TSG) Adenomatous Polyposis Coli ( APC ) is a hallmark event in colorectal cancers. Since it is not possible to directly target a TSG, no treatment options are available for these patients. Here, we identify SRP19 and the protein secretion machinery as a unique vulnerability in cancers with heterozygous APC loss. SRP19 is located 15 kb from APC and is almost always codeleted in these tumors. Heterozygous APC/SRP19 loss leads to lower levels of SRP19 mRNA and protein. Consequently, cells with APC/SRP19 loss are vulnerable to partial suppression of SRP19 . Moreover, we show that SRP19 is rate limiting for the formation of the Signal Recognition Particle, a complex that mediates ER-protein translocation, and thus, heterozygous SRP19 loss leads to less protein secretion and higher levels of ER-stress. As a result, low-dose arsenic trioxide induces ER-stress and inhibits proliferation in cultured cell lines and animal models. Our work identifies a strategy to treat cancers with APC deletion and provides a framework for identifying and translating vulnerabilities associated with loss of a TSG.
Advancing Nitrile-Aminothiol Strategy for Dual and Sequential Bioconjugation Varsha J. Thombare, Yimin Wu, Kavya Pamulapati, Meiling Han, Julien Tailhades, et al. Chemistry A European Journal, 2024 Nitrile‐aminothiol conjugation (NATC) stands out as a promising biocompatible ligation technique due to its high chemo‐selectivity. Herein we investigated the reactivity and substrate scope of NAT conjugation chemistry, thus developing a novel pH dependent orthogonal NATC as a valuable tool for chemical biology. The study of reaction kinetics elucidated that the combination of heteroaromatic nitrile and aminothiol groups led to the formation of an optimal bioorthogonal pairing, which is pH dependent. This pairing system was effectively utilized for sequential and dual conjugation. Subsequently, these rapid (≈1 h) and high yield (>90 %) conjugation strategies were successfully applied to a broad range of complex biomolecules, including oligonucleotides, chelates, small molecules and peptides. The effectiveness of this conjugation chemistry was demonstrated by synthesizing a fluorescently labelled antimicrobial peptide‐oligonucleotide complex as a dual conjugate to imaging in live cells. This first‐of‐its‐kind sequential NATC approach unveils unprecedented opportunities in modern chemical biology, showcasing exceptional adaptability in rapidly creating structurally complex bioconjugates. Furthermore, the results highlight its potential for versatile applications across fundamental and translational biomedical research.
Reassignment of the Structure of a Tryptophan-Containing Cyclic Tripeptide Produced by the Biarylitide Crosslinking Cytochrome P450blt Laura J. Coe, Yongwei Zhao, Leo Padva, Angus Keto, Ralf Schittenhelm, et al. Chemistry A European Journal, 2024 The structure of the sidechain crosslinked Tyr‐Leu‐Trp peptide produced by the biarylitide crosslinking cytochrome P450Blt from Micromonospora sp. MW‐13 has been reanalysed by a series of NMR, computational and isotope labelling experiments and shown to contain a C−N rather than a C−O bond. Additional in vivo experiments using such a modified peptide show there is a general tolerance of biarylitide crosslinking P450 enzymes for histidine to tryptophan mutations within their minimal peptide substrate sequences despite the lack of such residues noted in natural biarylitide gene clusters. This work further highlights the impressive ability of P450s from biarylitide biosynthesis pathways to act as biocatalysts for the formation of a range of sidechain crosslinked tripeptides.
An Engineered Biarylitide Cross-Linking P450 from RiPP Biosynthesis Generates Alternative Cyclic Peptides Maxine Treisman, Laura Coe, Yongwei Zhao, Vishnu Mini Sasi, Jemma Gullick, et al. Organic Letters, 2024 Cytochrome-P450-mediated cross-linking of ribosomally encoded peptides (RiPPs) is rapidly expanding and displays great potential for biocatalysis. Here, we demonstrate that active site engineering of the biarylitide cross-linking enzyme P450Blt enables the formation of His-X-Tyr and Tyr-X-Tyr cross-linked peptides, thus showing how such P450s can be further exploited to produce alternate cyclic tripeptides with controlled cross-linking states.
