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: | 13-三月-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 |
顯示於類別: | 期刊論文 |