In Vitro Reconstitution of OxyA Enzymatic Activity Clarifies Late Steps in Vancomycin Biosynthesis. Author Clarissa Forneris, Seyma Ozturk, Marcus Gibson, Erik Sorensen, Mohammad Seyedsayamdost Publication Year 2017 Type Journal Article Abstract Studies on the biosynthesis of glycopeptide antibiotics have provided many insights into the strategies that Nature employs to build architecturally strained molecules. A key structural feature of vancomycin, the founding member of this class, is a set of three aromatic cross-links that are introduced via yet unknown mechanisms. Previous reports have identified three cytochrome P450 enzymes involved in this process and demonstrated enzymatic activity for OxyB, which installs the first aromatic cross-link. However, the activities of the remaining two P450 enzymes have not been recapitulated. Herein, we show that OxyA generates the second bis-aryl ether bond in vancomycin and that it exhibits strict substrate specificity toward the chlorinated, OxyB-cross-linked product. No OxyA product is detected with the unchlorinated substrate. Together with previous results, these data suggest that chlorination occurs after OxyB- but before OxyA-catalyzed cross-link formation. Our results have important implications for the chemo-enzymatic synthesis of vancomycin and its analogs. Keywords Anti-Bacterial Agents, Substrate Specificity, Biosynthetic Pathways, Actinomycetales, Cytochrome P-450 Enzyme System, Halogenation, Vancomycin Journal ACS Chem Biol Volume 12 Issue 9 Pages 2248-2253 Date Published 2017 Sep 15 ISSN Number 1554-8937 DOI 10.1021/acschembio.7b00456 Alternate Journal ACS Chem Biol PMCID PMC5617736 PMID 28696669 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML