Three Self‐resistance Modifications Discovered in Nucleoside Antibiotic Muraymycin

Abstract

BackgroundMuraymycins, discovered by Wyeth Research in 2002, are lipopeptidyl nucleoside antibiotics that inhibit the enzyme bacterial translocase I involved in peptidoglycan cell wall. Muraymycin exhibits good activities against gram‐positive bacteria and excellent activity against a permeabilized E. coli. However, the self‐resistance mechanism during muraymycin biosynthesis has not been elucidated.ObjectivesOur long‐term research goal is to delineate the biosynthetic pathway of muraymycin in order to develop more active analogues through chemo‐enzymatic synthesis and mutasynthesis. Our current goal is to study the self‐resistance mechanism during muraymycin biosynthesis to assist structure–activity relationship (SAR) study of muraymycins.MethodsTo interrogate the self‐resistance mechanism during muraymycin biosynthesis, we analyzed its biosynthetic gene cluster. Two genes, mur28 and mur29 that potentially catalyzed the resistant modifications were cloned and expressed. We also cultured muraymycin‐producing strain to isolate resistant modified muraymycins. LC‐MS and NMR analysis was utilized to determine the resistant modified products.ResultsMur28 and Mur29 were functionally assigned in vitro as an ATP‐dependent phosphotransferase and an ATP‐dependent nucleotidyltransferase, both modifying the 3″‐OH of muraymycins. Interestingly, from single‐substrate kinetic analyses, we discovered that Mur28 preferentially phosphorylates a hypothetical biosynthetic precursor of muraymycin: a GlyU‐ADR disaccharide, while Mur29 preferentially adenylates muraymycin. Besides the two resistant modification products, a new muraymycin acetylaed at the 5″‐amine of the ADR moiety was isolated from muraymycin‐producing strain. The adenylated, phosphorylated and acetylated products have significantly reduced MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins.ConclusionPhosphorylation, nucleotidylation and acetylation, are three types of resistant modifications that are often found as mechanisms of resistance to antibiotics, most notably aminoglycoside antibiotics. Here, we first discovered all these three types of resistant modifications in the nucleoside antibiotic muraymycin. Among these, Mur28 serves as complementary self‐resistance mechanism, with a distinct temporal order during muraymycin biosynthesis.Support or Funding InformationThis work was supported by the National Institutes of Health (grant AI087849).TOCFigure 1