Characterization of the Stereoselective P450 Enzyme BotCYP Enables the <i>In Vitro</i> Biosynthesis of the Bottromycin Core Scaffold

Abstract

Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant <i>Staphylococcus aureus</i>. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot <i>in vitro</i> production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.