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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

Transformation of LBS to BS is significantly affected by fermentation conditions.(A) Schematic representation of transformation of LBS to BS; (B) HPLC analysis of metabolites produced by S. lividans WJ2 which was engrafted with the expanded BS biosynthetic gene cluster in different fermentations. BS is abbreviated for the blasticidin S, LBS is for leucylblasticidin S. The ratio between LBS to BS varied from 2:9 to 9:1 in different fermentations.
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f1: Transformation of LBS to BS is significantly affected by fermentation conditions.(A) Schematic representation of transformation of LBS to BS; (B) HPLC analysis of metabolites produced by S. lividans WJ2 which was engrafted with the expanded BS biosynthetic gene cluster in different fermentations. BS is abbreviated for the blasticidin S, LBS is for leucylblasticidin S. The ratio between LBS to BS varied from 2:9 to 9:1 in different fermentations.

Mentions: In studies of blasticidin S, demethylblasticidin S (DBS)21 and leucylblasticidin S (LBS)22 were isolated as the intermediates in the broth of S. griseochromogenes when the pH of the culture medium was kept below 4. However, LBS was later isolated from Streptomyces sp. SCC 1785 and S. griseochromogenes under the normal laboratory conditions2324. Addition of leucine to C-13 of the beta-amino group of BS was expected as a new self-resistance mechanism because the antibiotic activity of LBS against indicator strain Bacillus circulans was decreased by 20 folds compared to BS23. LBS was suggested as the direct precursor for BS biosynthesis as the washed cells22 or cell lysate23 of S. griseochromogenes can convert LBS into BS (Fig. 1A). 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.


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)

Transformation of LBS to BS is significantly affected by fermentation conditions.(A) Schematic representation of transformation of LBS to BS; (B) HPLC analysis of metabolites produced by S. lividans WJ2 which was engrafted with the expanded BS biosynthetic gene cluster in different fermentations. BS is abbreviated for the blasticidin S, LBS is for leucylblasticidin S. The ratio between LBS to BS varied from 2:9 to 9:1 in different fermentations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Transformation of LBS to BS is significantly affected by fermentation conditions.(A) Schematic representation of transformation of LBS to BS; (B) HPLC analysis of metabolites produced by S. lividans WJ2 which was engrafted with the expanded BS biosynthetic gene cluster in different fermentations. BS is abbreviated for the blasticidin S, LBS is for leucylblasticidin S. The ratio between LBS to BS varied from 2:9 to 9:1 in different fermentations.
Mentions: In studies of blasticidin S, demethylblasticidin S (DBS)21 and leucylblasticidin S (LBS)22 were isolated as the intermediates in the broth of S. griseochromogenes when the pH of the culture medium was kept below 4. However, LBS was later isolated from Streptomyces sp. SCC 1785 and S. griseochromogenes under the normal laboratory conditions2324. Addition of leucine to C-13 of the beta-amino group of BS was expected as a new self-resistance mechanism because the antibiotic activity of LBS against indicator strain Bacillus circulans was decreased by 20 folds compared to BS23. LBS was suggested as the direct precursor for BS biosynthesis as the washed cells22 or cell lysate23 of S. griseochromogenes can convert LBS into BS (Fig. 1A). 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.

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