標題: Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity
作者: Kang, Chia-Yu
Huang, I-Hsiu
Chou, Chi-Chi
Wu, Tsai-Yu
Chang, Jyun-Cyuan
Hsiao, Yu-Yuan
Cheng, Cheng-Hsuan
Tsai, Wei-Jiun
Hsu, Kai-Cheng
Wang, Shuying
生物科技學系
生物資訊及系統生物研究所
分子醫學與生物工程研究所
Department of Biological Science and Technology
Institude of Bioinformatics and Systems Biology
Institute of Molecular Medicine and Bioengineering
關鍵字: enzyme catalysis;fluorescence resonance energy transfer (FRET);protein structure;substrate specificity;protein chemistry;protein purification;protein sorting;Clostridium difficile;crystal structure;cysteine transpeptidase;sortase B;substrate specificity
公開日期: 13-Mar-2020
摘要: Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus. The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB?substrate complexes. Furthermore, we also demonstrated that residues 163?168 located on the ?6/?7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.
URI: http://dx.doi.org/10.1074/jbc.RA119.011322
http://hdl.handle.net/11536/154408
ISSN: 0021-9258
DOI: 10.1074/jbc.RA119.011322
期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
Volume: 295
Issue: 11
起始頁: 3734
結束頁: 3745
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