Chen, Jia-LiangWang, XiaoYang, FengCao, ChanOtting, GottfriedSu, Xun-Cheng2018-10-182018-10-180044-8249http://hdl.handle.net/1885/148493Enzyme 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 sortase A (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.application/pdf© 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim. http://www.sherpa.ac.uk/romeo/issn/0044-8249/..."author can archive post-print (ie final draft post-refereeing). 12 months embargo" from SHERPA/RoMEO site (as at 18/10/18). This is the pre-peer reviewed version of the following article: [Chen, Jia‐Liang, et al. "3D structure determination of an unstable transient enzyme intermediate by paramagnetic NMR spectroscopy." Angewandte Chemie International Edition 55.44 (2016): 13744-13748.], which has been published in final form at [https://dx.doi.org/10.1002/anie.201606223]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versionsnmr spectroscopyenzyme catalysisprotein dynamicsprotein structurestransient intermediatesaminoacyltransferasesbacterial proteinscysteine endopeptidasesmodels, molecularmolecular structureprotein conformationstaphylococcus aureusnuclear magnetic resonance, biomolecular201610.1002/anie.201606223