Investigations of valanimycin biosynthesis: elucidation of the role of seryl-tRNA

Abstract

The antibiotic valanimycin is a naturally occurring azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from l-valine and l-serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryl-tRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester O-seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an aminoacyl-tRNA in an antibiotic biosynthetic pathway.

Fig. 1.

Biosynthetic pathway for the antibiotic valanimycin. The pathway proceeds from l-valine and l-serine through the intermediacy of isobutylhydroxylamine.

Fig. 2.

Thin-layer chromatographic analysis of incubations with cell-free extracts of S. viridifaciens prepared from the wild type and four different mutants. Incubations were carried out at 30°C in a 100-μl volume for 16 h in the presence of 5 mM ATP, 50 μM l-[U-¹⁴C]serine, 0.5 mM isobutylhydroxylamine, 2 μg of VlmL, and 4 mg of total E. coli tRNA as described in Materials and Methods. At the end of the incubation period, a 2-μl aliquot of each incubation mixture was spotted onto a cellulose thin-layer plate, and the plate was developed with 1-butanol:acetic acid:water (25:4:10). The radioactive products were then visualized by autoradiography. R_f values are displayed for the two fastest-moving products. Seryl-tRNA remains at the baseline.

Fig. 3.

Thin-layer chromatographic analysis of incubations with VlmA and VlmL. Incubations were carried out at 30°C in a 100-μl volume over a period ranging from 0.5 to 24 h in the presence of 10 mM ATP, 0.1 mM l-[U-¹⁴C]serine, 0.75 mM isobutylhydroxylamine, 2 μg of VlmL, 10 μg of VlmA, and 4 mg of total E. coli tRNA as described in Materials and Methods. At the end of the incubation period, a 2-μl aliquot of each incubation mixture was spotted onto a cellulose thin-layer plate and the plate was developed with 1-butanol:acetic acid:water (25:4:10). The radioactive products were then visualized by autoradiography. The R_f values for the amide and ester are given in parentheses. Seryl-tRNA remains at the baseline.

Fig. 4.

Synthesis of N-(l-seryl)-isobutylhydroxylamine (8) and O-(l-seryl)-isobutylhydroxylamine (9). The amide 8 and the ester 9 were each synthesized in four steps from N-Boc-l-serine (1) as described in SI Text.

Fig. 5.

Probable mechanism for reaction catalyzed by VlmA. The reaction is postulated to proceed by nucleophilic attack of the hydroxyl group of isobutylhydroxylamine on the ester linkage of l-seryl-tRNA, resulting in the formation of O-(l-seryl)-isobutylhydroxylamine (9). The ester 9 was found to rearrange spontaneously to the amide N-(l-seryl)-isobutylhydroxylamine (8).

Fig. 6.

Hypothetical mechanisms for conversion of ester 9 into valanimycin. The most plausible mechanisms involve oxidation of 9 to the imine 10 or the N-hydroxy compound 11. The imine 10 or the N-hydroxy compound 11 could then be converted to the azo compound 13 in a manner described in greater detail in Results and Discussion.