3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy

• Published in: Angewandte Chemie International Edition 2016-10, Vol.55 (44), p.13744-13748
• Main Authors: Chen, Jia-Liang, Wang, Xiao, Yang, Feng, Cao, Chan, Otting, Gottfried, Su, Xun-Cheng
• Format: Article
• Language: English
• Subjects: Abundance; Catalysis; Constraints; Cysteine; enzyme catalysis; Enzymes; Equilibrium conditions; Hydrolysis; Intermediates; Magnetic resonance spectroscopy; NMR; NMR spectroscopy; Nuclear magnetic resonance; Plastic properties; Plasticity; protein dynamics; protein structures; Resonance; Spectrum analysis; transient intermediates
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201606223

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. Now you see it: 3D structures of low‐abundance transient enzyme intermediates can be determined from pseudocontact shifts (PCSs) measured by NMR spectroscopy. The method is demonstrated with the unstable thioester intermediate formed between Staphylococcus aureus sortase A and the peptide substrate.