Self-resistance during muraymycin biosynthesis: A complementary nucleotidyltransferase and phosphotransferase with identical modification sites and distinct temporal order

Abstract

Muraymycins are antibacterial natural products from Streptomyces spp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5=-C-glycyluridine (GlyU) appended to a 5-amino-5-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assigned in vitro as a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as co-substrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.