Self-Resistance During Muraymycin Biosynthesis: A Complementary Nucleotidyltransferase and Phosphotransferase with Identical Modification Sites and Distinct Temporal Order [PublishAheadOfPrint]

Muraymycins are antibacterial natural products from Streptomyces sp. that inhibit translocase I (MraY) involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5'-C-glycyluridine (GlyU) that is appended with 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, that both modify 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 analysis revealed Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetic biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced MraY inhibitory activity (170- and 51-fold, respectively) and reduced antibacterial activity when compared with the respective unmodified muraymycin. 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.

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