Abstract
The peptidyl nucleoside blasticidin S (BS) isolated from Streptomyces griseochromogenes was the first non-mercurial fungicide used on a large scale to prevent rice blast. In the biosynthesis of BS, leucylblasticidin S (LBS) was suggested as the penultimate metabolite with 20-fold less inhibitory activity than the final product BS. Incomplete conversion of LBS to BS at a variable efficiency ranging from 10% to 90% was observed either in the native strain S. griseochromogenes or a heterologous producer Streptomyces lividans WJ2. In this study, we determined that maturation of BS from LBS is not a spontaneous process but is governed by a standalone peptidase PepN, which hydrolyzes LBS in a pH-sensitive way with most appropriate of pH 7~8 but is inactive when the pH is below 5 or above 10. PepN1 and PepN2, two neighboring PepN homologs from Streptomyces lividans were purified in E. coli but displayed ca.100-fold difference in LBS hydrolytic activity. Overexpression of pepN1 in WJ2 enhanced BS yield by 100% and lowered the ratio of LBS to BS from 2:1 to 2:3. This work presents the expansion of the biological role for PepN in antibiotic maturation and the first report of hydrolysis of beta amide linkage by this conserved enzyme.
Highlights
Blasticidin S (BS) is a nucleoside analog consisting of a cytosine linked to a dideoxyhexose and bonded to a modified arginine
Removal of the leucyl group from Leucylblasticidin S was proposed as the final step in BS biosynthesis[23]
Heterologous expression of blasticidin S biosynthetic gene cluster in Streptomyces lividans occasionally produces the mixture of leucylblasticidin S (LBS) and BS
Summary
Blasticidin S (BS) is a nucleoside analog consisting of a cytosine linked to a dideoxyhexose and bonded to a modified arginine. Blasticidin S exerts its inhibitory activity by interfering with ribosomal protein synthesis in host cells. The first group featured by BSD or BSR encodes blasticidin S deaminase[8,9,10,11,12] that converts BS to non-toxic deaminohydroxyblasticidin S. This group of resistance genes along with BS are widely used in the selection of the transformed cells, in eukaryotic transgenic studies[13,14,15]. It is of particular interest to know where this conversion occurs to avoid self-destruction of the host by the generated BS, as well as identifying the protein responsible for this hydrolysis in vivo
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