The glycopeptide antibiotics, exemplified by teicoplanin and vancomycin, are complex secondary metabolites that are found in the last line of medical defence against resistant Gram-positive bacteria. The biosynthesis of these molecules centres on the actions and direct interplay of the peptide-producing non-ribosomal peptide synthetase and the Oxy proteins – members of the Cytochrome P450 superfamily of mono-oxygenases – that are responsible for the oxidative phenolic or aryl crosslinking of the aromatic side chains of the precursor peptide to afford the corresponding glycopeptide antibiotic aglycones in their active conformation.
With the rise in bacterial resistance against even the glycopeptide antibiotics, the need for new active compounds is ever increasing. Given that the oxidative crosslinking reactions performed by the Oxy proteins are not only crucial for antibiotic activity but also represent a significant challenge in the chemical synthesis of glycopeptide antibiotics, we have concentrated on understanding this process. In order to achieve this, we have developed an improved synthetic route to glycopeptide antibiotic precursor peptides using Fmoc-chemistry that also enables enzymatic conjugation of these peptides through a phosphopantheine linker: this has enabled us to show that the crucial glycopeptide antibiotic crosslinking reactions occur whilst the peptide is covalently bound to the non-ribosomal peptide synthetase itself, with the recruitment of the oxygenase enzymes mediated by a specific domain in the biosynthetic machinery.
This presentation will highlight recent results from our investigations, which include elucidation of the mode of interaction of Oxy proteins with the non-ribosomal peptide synthetase machinery as well as the in vitro characterisation of crosslinking reactions performed by previously uncharacterised Oxy proteins. The potential for exploitation of these enzymes in the sidechain crosslinking of novel peptides will also be addressed.