Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

Chia Yu Kang, I. Hsiu Huang, Chi Chi Chou, Tsai Yu Wu, Jyun Cyuan Chang, Yu Yuan Hsiao, Cheng Hsuan Cheng, Wei Jiun Tsai, Kai Cheng Hsu, Shuying Wang

研究成果: Article

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 β6/β7 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.

原文English
頁(從 - 到)3734-3745
頁數12
期刊Journal of Biological Chemistry
295
發行號11
DOIs
出版狀態Published - 2020 三月 13

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Kang, C. Y., Huang, I. H., Chou, C. C., Wu, T. Y., Chang, J. C., Hsiao, Y. Y., Cheng, C. H., Tsai, W. J., Hsu, K. C., & Wang, S. (2020). Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity. Journal of Biological Chemistry, 295(11), 3734-3745. https://doi.org/10.1074/jbc.RA119.011322