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The standalone aminopeptidase PepN catalyzes the maturation of blasticidin S from leucylblasticidin S.

Yu G, Li L, Liu X, Liu G, Deng Z, Zabriskie MT, Jiang M, He X - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 (China).

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.

No MeSH data available.


Related in: MedlinePlus

HPLC analysis of the reaction products for incubation of LBS with the CFEs of mutants of peptidase genes.Only the pepN (∆9) mutant lost all LBS hydrolytic activity while the other four mutants retained the same efficacy in LBS hydrolysis as the wild type.
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f4: HPLC analysis of the reaction products for incubation of LBS with the CFEs of mutants of peptidase genes.Only the pepN (∆9) mutant lost all LBS hydrolytic activity while the other four mutants retained the same efficacy in LBS hydrolysis as the wild type.

Mentions: The five peptidase genes were then disrupted in E. coli BW25113, an E. coli derivative facilitating homologous recombination between linear DNA fragment and chromosome. The CFE of the five mutants were assayed for LBS hydrolytic activity. Only the pepN mutant lost all LBS hydrolytic activity while the other four mutants retained the same efficacy in LBS hydrolysis as the wild type (Fig. 4). This result demonstrates that PepN is responsible for LBS hydrolysis in E. coli.


The standalone aminopeptidase PepN catalyzes the maturation of blasticidin S from leucylblasticidin S.

Yu G, Li L, Liu X, Liu G, Deng Z, Zabriskie MT, Jiang M, He X - Sci Rep (2015)

HPLC analysis of the reaction products for incubation of LBS with the CFEs of mutants of peptidase genes.Only the pepN (∆9) mutant lost all LBS hydrolytic activity while the other four mutants retained the same efficacy in LBS hydrolysis as the wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664946&req=5

f4: HPLC analysis of the reaction products for incubation of LBS with the CFEs of mutants of peptidase genes.Only the pepN (∆9) mutant lost all LBS hydrolytic activity while the other four mutants retained the same efficacy in LBS hydrolysis as the wild type.
Mentions: The five peptidase genes were then disrupted in E. coli BW25113, an E. coli derivative facilitating homologous recombination between linear DNA fragment and chromosome. The CFE of the five mutants were assayed for LBS hydrolytic activity. Only the pepN mutant lost all LBS hydrolytic activity while the other four mutants retained the same efficacy in LBS hydrolysis as the wild type (Fig. 4). This result demonstrates that PepN is responsible for LBS hydrolysis in E. coli.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 (China).

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.

No MeSH data available.


Related in: MedlinePlus