Dataset Information

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of ?-Hydroxylation during Glycopeptide Antibiotic Biosynthesis.

ABSTRACT: ?-Hydroxylation plays an important role in the nonribosomal peptide biosynthesis of many important natural products, including bleomycin, chloramphenicol, and the glycopeptide antibiotics (GPAs). Various oxidative enzymes have been implicated in such a process, with the mechanism of incorporation varying from installation of hydroxyl groups in amino acid precursors prior to adenylation to direct amino acid oxidation during peptide assembly. In this work, we demonstrate the in vitro utility and scope of the unusual nonheme diiron monooxygenase CmlA from chloramphenicol biosynthesis for the ?-hydroxylation of a diverse range of carrier protein bound substrates by adapting this enzyme as a non-native trans-acting enzyme within NRPS-mediated GPA biosynthesis. The results from our study show that CmlA has a broad substrate specificity for modified phenylalanine/tyrosine residues as substrates and can be used in a practical strategy to functionally cross complement compatible NRPS biosynthesis pathways in vitro.

SUBMITTER: Kaniusaite M

PROVIDER: S-EPMC6929969 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Publications

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of β-Hydroxylation during Glycopeptide Antibiotic Biosynthesis.

Kaniusaite Milda M Goode Robert J A RJA Schittenhelm Ralf B RB Makris Thomas M TM Cryle Max J MJ

ACS chemical biology 20191210 12

β-Hydroxylation plays an important role in the nonribosomal peptide biosynthesis of many important natural products, including bleomycin, chloramphenicol, and the glycopeptide antibiotics (GPAs). Various oxidative enzymes have been implicated in such a process, with the mechanism of incorporation varying from installation of hydroxyl groups in amino acid precursors prior to adenylation to direct amino acid oxidation during peptide assembly. In this work, we demonstrate the <i>in vitro</i> utilit ...[more]

PMID: 31774267

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Project description:Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains - known as condensation domains - is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

Dataset Information

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of ?-Hydroxylation during Glycopeptide Antibiotic Biosynthesis.

Publications

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of β-Hydroxylation during Glycopeptide Antibiotic Biosynthesis.

Similar Datasets

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