<jats:title>ABSTRACT</jats:title> <jats:p> The Trojan horse antibiotic albomycin, produced by <jats:italic>Streptomyces</jats:italic> 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 <jats:italic>Staphylococcus aureus</jats:italic> , with a 50% inhibitory concentration (IC <jats:sub>50</jats:sub> ) of ∼8 nM. In the albomycin-producing <jats:italic>Streptomyces</jats:italic> sp., a bacterial SerRS homolog (Alb10) was found to be encoded in a biosynthetic gene cluster in addition to another <jats:italic>s</jats:italic> <jats:italic>erRS</jats:italic> gene ( <jats:italic>serS1</jats:italic> ) 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 <jats:italic>Streptomyces</jats:italic> species. We genetically and biochemically characterized the two genes and the proteins encoded. Both genes were able to complement a temperature-sensitive <jats:italic>serS</jats:italic> mutant of <jats:italic>Escherichia coli</jats:italic> and allowed growth at a nonpermissive temperature. <jats:italic>serS2</jats:italic> 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 <jats:italic> K <jats:sub>m</jats:sub> </jats:italic> 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 <jats:sub>50</jats:sub> 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 <jats:italic>Streptomyces</jats:italic> genomes for duplicated aminoacyl-tRNA synthetase genes could provide a novel approach for the identification of natural products targeting aminoacyl-tRNA synthetases.