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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 product by incubating LBS with the cell free extracts (CFE) of Streptomyces griseochromogenes, Saccharomyces sake, E. coli DH10B.All of them were found to convert LBS into BS. Boiled CFE of DH10B was used as negative control.
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f2: HPLC analysis of the reaction product by incubating LBS with the cell free extracts (CFE) of Streptomyces griseochromogenes, Saccharomyces sake, E. coli DH10B.All of them were found to convert LBS into BS. Boiled CFE of DH10B was used as negative control.

Mentions: The washed cells of S. griseochromogenes, the BS native producer, were reported to hydrolyze LBS into BS22. We repeated this experiment using the cells of S. lividans WJ2 (harboring the BS gene cluster) and its parent strain S. lividans HXY16 (without the BS gene cluster) as the negative control. Surprisingly, the cells of both strains are capable of hydrolyzing LBS to BS in similar effective way. We further found that cells from E. coli DH10B and Saccharomyces sake are also able to accomplish this process. The cell free extracts of these strains were all found to possess LBS hydrolytic activity as well (Fig. 2). However, the boiled CFE of E. coli DH10B cannot perform this process, demonstrating that LBS hydrolysis is not a spontaneous process and that there is a conserved hydrolase gene in charge of the maturation of LBS into active BS.


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 product by incubating LBS with the cell free extracts (CFE) of Streptomyces griseochromogenes, Saccharomyces sake, E. coli DH10B.All of them were found to convert LBS into BS. Boiled CFE of DH10B was used as negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: HPLC analysis of the reaction product by incubating LBS with the cell free extracts (CFE) of Streptomyces griseochromogenes, Saccharomyces sake, E. coli DH10B.All of them were found to convert LBS into BS. Boiled CFE of DH10B was used as negative control.
Mentions: The washed cells of S. griseochromogenes, the BS native producer, were reported to hydrolyze LBS into BS22. We repeated this experiment using the cells of S. lividans WJ2 (harboring the BS gene cluster) and its parent strain S. lividans HXY16 (without the BS gene cluster) as the negative control. Surprisingly, the cells of both strains are capable of hydrolyzing LBS to BS in similar effective way. We further found that cells from E. coli DH10B and Saccharomyces sake are also able to accomplish this process. The cell free extracts of these strains were all found to possess LBS hydrolytic activity as well (Fig. 2). However, the boiled CFE of E. coli DH10B cannot perform this process, demonstrating that LBS hydrolysis is not a spontaneous process and that there is a conserved hydrolase gene in charge of the maturation of LBS into active BS.

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