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 spp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5'--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 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 cosubstrates 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.

Theoretical Study of Intermolecular Interactions between Critical Residues of Membrane Protein MraYAAand Promising Antibiotic Muraymycin D2

“… 7 , 9 If the peptide of muraymycins is l -leucine and there is a hydroxyl group in the 2″-position of the ADR moiety, muraymycin will be classified as muraymycin D2 (MD2) ( Figure 1 ). 9 …”

Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics

“…LCMS analysis of the new peak yielded an (M + H) + ion at m/z = 449.1463 (Figure 8C), which is consistent with the molecular formula for ADR-GlyU disaccharide ( 17a ) [expected (M + H) + ion at m/z = 449.1442 for C 16 H 24 N 4 O 11 ]. To simplify the analytical identification of the product, 17a was prepared by chemical synthesis (Figures S8-S12); 30 analysis of the Mur19-catalyzed reaction revealed the new peak co-eluted with, and had identical UV and MS spectroscopic properties to, authentic 17a . The data are therefore consistent with the functional assignment of Mur19 as a 12 : 15a 5-amino-5-deoxyribosyltransferase.…”

“…The stereoselective synthesis of the disaccharide 17a followed a previously described procedure, 30 which is based on the methodology first reported by Hirano et al 4,34,35 1 H NMR (500 MHz, D 2 O) δ 7.74 (d, J = 8.0 Hz, 1H), 5.87 (d, J = 8.0 Hz, 1H), 5.81 (d, J = 2.7 Hz, 1H), 5.17 (s, 1H), 4.50 (br s, 1H), 4.35 (dd, J = 5.0, 2.7 Hz, 1H), 4.18–4.26 (m, 2H), 4.08–4.17 (m, 3H), 3.88 (br s, 1H), 3.25–3.38 (m, 1H), 3.06–3.15 (m, 1H); 13 C NMR (126 MHz, D 2 O) δ 167.4, 152.3, 141.7, 109.0, 102.0, 91.0, 84.7, 79.1, 77.2, 75.0, 73.1, 72.0, 69.3, 57.2, 42.5; HRMS (ESI/Q-TOF) m/z: [M + H] + Calcd for C 16 H 25 N 4 O 11 449.1520; Found 449.1462; IR (ATR) ν 3139, 2935, 2365, 2336, 1703, 1684, 1627, 1510, 1470, 1395, 1272, 1197, 1121, 1051, 1005, 819, 796, 720; UV (H 2 O) λ max (log ε) 202 (2.13), 262 (2.10); mp 172 °C (decomposition); TLC R f 0.05–0.16 (5:2:1 i -PrOH-H 2 O-AcOH as saturated NaCl solution); [α] D 20 +3.8 ( c 0.90, H 2 O).…”

Characterization of the Self-Resistance Mechanism to Dityromycin in the Streptomyces Producer Strain

“…Resistance genes are often carried by the same gene cluster of the antibiotic biosynthetic enzymes, and their expression is temporally coupled. Indeed, antibiotic producer cells need to express resistance determinants prior to, or concomitantly with, synthesis of the antibiotic molecule (26, 27). In recent years, the analysis of several genomes suggested that microorganisms produce only a fraction of the secondary metabolites that they encode, and new approaches have been used to identify biosynthetic gene clusters whose expression can be induced.…”