Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes

• DOI: 10.1038/nchembio.1385
• Publication Date: 2013-11-17T18:27:24Z
• Authors: Raoul G Rosenthal; Marc-Olivier Ebert; Patrick Kiefer; Dominik M Peter; Julia A Vorholt; Tobias J Erb
• Keywords: Kinetics; 0303 health sciences; 03 medical and health sciences; Magnetic Resonance Spectroscopy; Catalysis; Mass Spectrometry; NADP; Enzymes

The pyridine nucleotides NADH and NADPH (NAD(P)H) are ubiquitous redox coenzymes that are present in all living cells. Although about 16% of all characterized enzymes use pyridine nucleotides as hydride donors or acceptors during catalysis, a detailed understanding of how the hydride is transferred between NAD(P)H and the corresponding substrate is lacking for many enzymes. Here we present evidence for a new mechanism that operates during enzymatic hydride transfers using crotonyl-CoA carboxylase/reductase (Ccr) as a case study. We observed a covalent ene intermediate between NADPH and the substrate, crotonyl-CoA, using NMR, high-resolution MS and stopped-flow spectroscopy. Preparation of the ene intermediate further allowed direct access to the catalytic cycle of other NADPH-dependent enzymes-including those from type II fatty acid biosynthesis-in an unprecedented way, suggesting that formation of NAD(P)H ene intermediates is a more general principle in catalysis.