期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 55, 期 44, 页码 13744-13748出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201606223
关键词
enzyme catalysis; NMR spectroscopy; protein dynamics; protein structures; transient intermediates
资金
- Major National Scientific Research Projects [2013CB910200, 2016YFA0501202]
- National Natural Science Foundation of China [21473095, 21273121]
- Australian Research Council [DP140100304]
Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three-dimensional (3D) structure of an unstable, low-abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortaseA (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site-specific cysteine-reactive paramagnetic tag that does not react with the active-site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non-equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.
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