Two Cryptic Self‐Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin

Abstract

Apramycin is aclinically promising aminoglycoside antibiotic (AGA). To date,m echanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps,i nw hich an ovel aminoglycoside phosphotransferase (AprU), ap utative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of ap seudotrisaccharide intermediate,w hose N-7' acetyl group is subsequently hydrolyzed by AprP.A prZ is located extracellularly where it removes the phosphate group from ap seudotetrasaccharide intermediate,leading to the maturation of apramycin. Intriguingly,7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in DaprU and DaprZ respectively exhibited significantly reduced antibacterial activities,i mplying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity.S ignificantly,t his study provides insight into the design of new generation AGAs to circumvent the emergence of drugresistant pathogens.

Conserved Mechanism of 2′-Phosphorylation-Aided Amide Ligation in Peptidyl Nucleoside Biosynthesis

“…The proposed mechanism involves phosphorylation of 2′-OH of a precursor nucleotide early in the pathway, which is essential for the activity of all subsequent AHA biosynthetic enzymes. Analogous cryptic phosphorylation has also been found in other nucleoside − and aminoglycoside natural products, suggesting the generality of this biosynthetic mechanism.…”

“…The results described above suggest that the amide ligation step in the nikkomycin and polyoxin pathways requires 2′-phosphorylation. While the identity of the enzyme responsible for the dephosphorylation of nikkomycin or polyoxin 2′-phosphate is currently unknown, a recent study of apramycin biosynthesis identified a promiscuous phosphatase (AprZ) as being responsible for dephosphorylating apramycin phosphate after the metabolite’s translocation to the periplasm. Although no such phosphatase is encoded in the nikkomycin or polyoxin pathway, it is possible that a similar phosphatase outside the biosynthetic gene cluster promiscuously dephosphorylates 2′-phosphorylated PNs once exported to the periplasm.…”

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics

“…Actually, enzyme-guided chemical modification of compound is one of the most widely existed strategies for antibiotic resistance including self-resistance involved in biosynthetic pathways. However, these modifications are usually limited by reversible group-transfer reactions acting on end-products, such as methylation, acylation, phosphorylation, glycosylation, amino-, or peptide-acylation and so on 1 , 36 . Rare cases employing reductive reaction include virginiamycin M1 reductase-catalyzed conversion of a ketone to alcohol 37 , a nitroreductase-mediated chloramphenicol inactivation and CytA-catalyzed dehydroxylation of cytorhodin 38 , 39 , as well as two recently-discovered fungal examples including a multistep reduction cascade in monasone naphthoquinone and a redox-cycle in A26771B biosynthesis 33 , 40 .…”

Monooxygenase LaPhzX is Involved in Self-Resistance Mechanisms during the Biosynthesis of N-Oxide Phenazine Myxin

“…Besides the mentioned self-resistance mechanisms in the biosynthesis of fungal macrolide antibiotic A26771B and naphthyridinomycin, an increasing number of AEMs involved in self-resistance cases have been reported in antibiotic biosynthesis. For example, apramycin biosynthesis proceeding through phosphorylation and acetylation with AEMs are two cryptic self-resistance mechanisms in Streptomyces tenebrarius . In addition, a tRNA-dependent aminoacyltransferase BlsK, which is responsible for leucyl transfer in the biosynthesis of Blasticidin S, has been demonstrated to be a new self-resistance mechanism in Streptomyces griseochromogenes .…”

“…For example, apramycin biosynthesis proceeding through phosphorylation and acetylation with AEMs are two cryptic self-resistance mechanisms in Streptomyces tenebrarius. 33 In addition, a tRNA-dependent aminoacyltransferase BlsK, which is responsible for leucyl transfer in the biosynthesis of Blasticidin S, has been demonstrated to be a new self-resistance mechanism in Streptomyces griseochromogenes. 34 However, the AEM-mediated self-resistance mechanisms employed in phenazine biosynthesis are poorly understood.…”