title: Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli.
abstract: Glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL) is widely used in peptide recovery and synthesis because of its unique substrate specificity. However, the mechanism underlying its specificity is still not thoroughly understood. In this study, the roles of the prosegment and key amino acids involved in the proteolytic activity of GSE-BL were investigated. Loss of the GSE-BL prosegment severely restricted enzymatic activity toward Z-Phe-Leu-Glu-pNA. A homologous model of GSE-BL revealed that it contains the catalytic triad "His47, Asp96 and Ser 167", which was further confirmed by site-directed mutagenesis. In vitro mutagenesis further indicated that Val2, Arg89 and His190 are essential for enzymatic activity toward Z-Phe-Leu-Glu-pNA. Moreover, the catalytic efficiency of Phe57Ala GSE-BL toward Z-Phe-Leu-Glu-pNA was 50% higher than that of the native mature GSE-BL. This is the first study to fully elucidate the key amino acids for proteolytic activity of GSE-BL. Mature GSE-BL could be obtained through self-cleavage alone when Lys at -1 position was replaced by Glu, providing a new strategy for the preparation of mature GSE-BL. This study yielded some valuable insights into the substrate specificity of glutamate-specific endopeptidase, establishing a foundation for broadening the applications of GSE-BL.
authors: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China; State Key Laboratory of Applied Microbiology, South China (The Ministry-Province Joint Development), Guangdong Institute of Microbiology, GuangZhou 510070, PR China.
authors: Wei
authors: W
authors: Ye
authors: Haiying
authors: H
authors: Wang
authors: Yi
authors: Y
authors: Ma
authors: Xiaochun
authors: X
authors: Luo
authors: Weimin
authors: W
authors: Zhang
authors: Jufang
authors: J
authors: Wang
authors: Xiaoning
authors: X
authors: Wang
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed
chemical_list: D018698:Glutamic Acid; D010450:Endopeptidases
keywords: Catalytic triad; Enzymatic activity; GSE-BI; GSE-BL; GSE-SG; Glutamate-specific endopeptidase from Bacillus licheniformis; Prosegment; Substrate binding pocket; V8-GSE; Z; carboxybenzyl; glutamate-specific endopeptidase from Bacillus intermedius; glutamate-specific endopeptidase from Bacillus licheniformis; glutamate-specific endopeptidase from Staphylococcus aureus V8; glutamate-specific endopeptidase from Streptomyces grieus; nitrophenylamine; pNA