Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974

Abstract

The Trojan horse antibiotic albomycin, produced by Streptomyces sp. strain ATCC 700974, contains a thioribosyl nucleoside moiety linked to a hydroxamate siderophore through a serine residue. The seryl nucleoside structure (SB-217452) is a potent inhibitor of seryl-tRNA synthetase (SerRS) in the pathogenic bacterium Staphylococcus aureus, with a 50% inhibitory concentration (IC(50)) of approximately 8 nM. In the albomycin-producing Streptomyces sp., a bacterial SerRS homolog (Alb10) was found to be encoded in a biosynthetic gene cluster in addition to another serRS gene (serS1) at a different genetic locus. Alb10, named SerRS2 herein, is significantly divergent from SerRS1, which shows high homology to the housekeeping SerRS found in other Streptomyces species. We genetically and biochemically characterized the two genes and the proteins encoded. Both genes were able to complement a temperature-sensitive serS mutant of Escherichia coli and allowed growth at a nonpermissive temperature. serS2 was shown to confer albomycin resistance, with specific amino acid residues in the motif 2 signature sequences of SerRS2 playing key roles. SerRS1 and SerRS2 are comparably efficient in vitro, but the K(m) of serine for SerRS2 measured during tRNA aminoacylation is more than 20-fold higher than that for SerRS1. SB-217452 was also enzymatically generated and purified by two-step chromatography. Its IC(50) against SerRS1 was estimated to be 10-fold lower than that against SerRS2. In contrast, both SerRSs displayed comparable inhibition kinetics for serine hydroxamate, indicating that SerRS2 was specifically resistant to SB-217452. These data suggest that mining Streptomyces genomes for duplicated aminoacyl-tRNA synthetase genes could provide a novel approach for the identification of natural products targeting aminoacyl-tRNA synthetases.