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- Title
3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy.
- Authors
Chen, Jia ‐ Liang; Wang, Xiao; Yang, Feng; Cao, Chan; Otting, Gottfried; Su, Xun ‐ Cheng
- Abstract
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 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.
- Subjects
INTERMEDIATES (Chemistry); NUCLEAR magnetic resonance spectroscopy; CATALYSIS; SORTASES; STAPHYLOCOCCUS aureus; CATALYTIC hydrolysis
- Publication
Angewandte Chemie, 2016, Vol 128, Issue 44, p13948
- ISSN
0044-8249
- Publication type
Article
- DOI
10.1002/ange.201606223