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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

SDS-PAGE analysis of the protein fractions eluted from MonoQ HR 5/5 column. “−” represents this fraction has no LBS hydrolysis activity.“ + ” represents positive in LBS hydrolysis. There are some comparatively thicker bands in the most active No.16 fraction, such as bands of approximate 95 kD and 66 kD indicated by arrows.
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f3: SDS-PAGE analysis of the protein fractions eluted from MonoQ HR 5/5 column. “−” represents this fraction has no LBS hydrolysis activity.“ + ” represents positive in LBS hydrolysis. There are some comparatively thicker bands in the most active No.16 fraction, such as bands of approximate 95 kD and 66 kD indicated by arrows.

Mentions: As shown in Fig. 2, E. coli, the most studied bacterium, displayed relatively high efficiency in LBS hydrolysis and was thus chosen as the start strain to clone LBS hydrolase gene. The CFE of E. coli were precipitated with different concentration of ammonium sulfate wherein the fraction precipitated by 40% (V/V) ammonium sulfate displayed high catalytic activity, and further subjected to sequential separation on HiTrap Q HP (GE Healthcare, 5 ml) and on MonoQ HR 5/5 column (GE Healthcare). The eluate fractions from No.15 till No.19 showed LBS hydrolysis activity. These fractions and the inactive fraction No. 14 were analyzed by gel electrophoresis. There are some comparatively thicker bands in the most active No.16 fraction, such as bands of approximate 95 kD and 66 kD (indicated by arrows in Fig. 3). The fractions of No.14 and No.16 were analyzed by LC-MS/MS for their protein compositions (Supplementary Figure S2). 296 specific proteins were identified in the fraction of No.16 wherein the five most abundant peptidases were chosen as the candidates for the LBS hydrolase (Table 1).


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)

SDS-PAGE analysis of the protein fractions eluted from MonoQ HR 5/5 column. “−” represents this fraction has no LBS hydrolysis activity.“ + ” represents positive in LBS hydrolysis. There are some comparatively thicker bands in the most active No.16 fraction, such as bands of approximate 95 kD and 66 kD indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: SDS-PAGE analysis of the protein fractions eluted from MonoQ HR 5/5 column. “−” represents this fraction has no LBS hydrolysis activity.“ + ” represents positive in LBS hydrolysis. There are some comparatively thicker bands in the most active No.16 fraction, such as bands of approximate 95 kD and 66 kD indicated by arrows.
Mentions: As shown in Fig. 2, E. coli, the most studied bacterium, displayed relatively high efficiency in LBS hydrolysis and was thus chosen as the start strain to clone LBS hydrolase gene. The CFE of E. coli were precipitated with different concentration of ammonium sulfate wherein the fraction precipitated by 40% (V/V) ammonium sulfate displayed high catalytic activity, and further subjected to sequential separation on HiTrap Q HP (GE Healthcare, 5 ml) and on MonoQ HR 5/5 column (GE Healthcare). The eluate fractions from No.15 till No.19 showed LBS hydrolysis activity. These fractions and the inactive fraction No. 14 were analyzed by gel electrophoresis. There are some comparatively thicker bands in the most active No.16 fraction, such as bands of approximate 95 kD and 66 kD (indicated by arrows in Fig. 3). The fractions of No.14 and No.16 were analyzed by LC-MS/MS for their protein compositions (Supplementary Figure S2). 296 specific proteins were identified in the fraction of No.16 wherein the five most abundant peptidases were chosen as the candidates for the LBS hydrolase (Table 1).

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