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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

pH is the key factor in regulating PepN activity in vitro and in vivo.(A) HPLC analysis of reaction products for purified PepN1 with excessive LBS at pH ranging from 5 to 9 for 5 minutes, the most appropriate pH for in vitro reaction at 7.5 and least active above 10 or below 6, PepN activity is significantly affected by pH. (B) The production of BS and LBS and their total amount by S. lividans WJ2 when the pH of the fermentation medium was pre-adjusted to 5, 6, 7, 8 and 9, the pH is adjusted after medium sterilization.
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f5: pH is the key factor in regulating PepN activity in vitro and in vivo.(A) HPLC analysis of reaction products for purified PepN1 with excessive LBS at pH ranging from 5 to 9 for 5 minutes, the most appropriate pH for in vitro reaction at 7.5 and least active above 10 or below 6, PepN activity is significantly affected by pH. (B) The production of BS and LBS and their total amount by S. lividans WJ2 when the pH of the fermentation medium was pre-adjusted to 5, 6, 7, 8 and 9, the pH is adjusted after medium sterilization.

Mentions: As in different fermentations, the proportion of LBS in the final metabolites varies, implying that PepN activity is affected by the changing medium conditions, particularly the pH value as it was affected very much by cell contamination or sterilizations process. PepN and PepN1 were then measured for their activity in hydrolysis of LBS at pH ranging from 5 to 9. PepN and PepN1 behave similarly at the same pH, and the pattern change of activity are also similar, namely with most appropriate pH for in vitro reaction at 7.5 and least activity above 10 or below 5 (Fig. 5A). These observation are consistent with that pH variation in the fermentation broth affected the LBS hydrolase activity (Fig. 5B) and thus determined the proportion of LBS to BS in the final metabolites.


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)

pH is the key factor in regulating PepN activity in vitro and in vivo.(A) HPLC analysis of reaction products for purified PepN1 with excessive LBS at pH ranging from 5 to 9 for 5 minutes, the most appropriate pH for in vitro reaction at 7.5 and least active above 10 or below 6, PepN activity is significantly affected by pH. (B) The production of BS and LBS and their total amount by S. lividans WJ2 when the pH of the fermentation medium was pre-adjusted to 5, 6, 7, 8 and 9, the pH is adjusted after medium sterilization.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: pH is the key factor in regulating PepN activity in vitro and in vivo.(A) HPLC analysis of reaction products for purified PepN1 with excessive LBS at pH ranging from 5 to 9 for 5 minutes, the most appropriate pH for in vitro reaction at 7.5 and least active above 10 or below 6, PepN activity is significantly affected by pH. (B) The production of BS and LBS and their total amount by S. lividans WJ2 when the pH of the fermentation medium was pre-adjusted to 5, 6, 7, 8 and 9, the pH is adjusted after medium sterilization.
Mentions: As in different fermentations, the proportion of LBS in the final metabolites varies, implying that PepN activity is affected by the changing medium conditions, particularly the pH value as it was affected very much by cell contamination or sterilizations process. PepN and PepN1 were then measured for their activity in hydrolysis of LBS at pH ranging from 5 to 9. PepN and PepN1 behave similarly at the same pH, and the pattern change of activity are also similar, namely with most appropriate pH for in vitro reaction at 7.5 and least activity above 10 or below 5 (Fig. 5A). These observation are consistent with that pH variation in the fermentation broth affected the LBS hydrolase activity (Fig. 5B) and thus determined the proportion of LBS to BS in the final metabolites.

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