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dc.contributor.authorKang, Chia-Yuen_US
dc.contributor.authorHuang, I-Hsiuen_US
dc.contributor.authorChou, Chi-Chien_US
dc.contributor.authorWu, Tsai-Yuen_US
dc.contributor.authorChang, Jyun-Cyuanen_US
dc.contributor.authorHsiao, Yu-Yuanen_US
dc.contributor.authorCheng, Cheng-Hsuanen_US
dc.contributor.authorTsai, Wei-Jiunen_US
dc.contributor.authorHsu, Kai-Chengen_US
dc.contributor.authorWang, Shuyingen_US
dc.date.accessioned2020-07-01T05:21:20Z-
dc.date.available2020-07-01T05:21:20Z-
dc.date.issued2020-03-13en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://dx.doi.org/10.1074/jbc.RA119.011322en_US
dc.identifier.urihttp://hdl.handle.net/11536/154408-
dc.description.abstractMost 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.en_US
dc.language.isoen_USen_US
dc.subjectenzyme catalysisen_US
dc.subjectfluorescence resonance energy transfer (FRET)en_US
dc.subjectprotein structureen_US
dc.subjectsubstrate specificityen_US
dc.subjectprotein chemistryen_US
dc.subjectprotein purificationen_US
dc.subjectprotein sortingen_US
dc.subjectClostridium difficileen_US
dc.subjectcrystal structureen_US
dc.subjectcysteine transpeptidaseen_US
dc.subjectsortase Ben_US
dc.subjectsubstrate specificityen_US
dc.titleFunctional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificityen_US
dc.typeArticleen_US
dc.identifier.doi10.1074/jbc.RA119.011322en_US
dc.identifier.journalJOURNAL OF BIOLOGICAL CHEMISTRYen_US
dc.citation.volume295en_US
dc.citation.issue11en_US
dc.citation.spage3734en_US
dc.citation.epage3745en_US
dc.contributor.department生物科技學系zh_TW
dc.contributor.department生物資訊及系統生物研究所zh_TW
dc.contributor.department分子醫學與生物工程研究所zh_TW
dc.contributor.departmentDepartment of Biological Science and Technologyen_US
dc.contributor.departmentInstitude of Bioinformatics and Systems Biologyen_US
dc.contributor.departmentInstitute of Molecular Medicine and Bioengineeringen_US
dc.identifier.wosnumberWOS:000527725300028en_US
dc.citation.woscount0en_US
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