Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent 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.
1] Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland. [2].
Raoul G
RG
Rosenthal
1] Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland. [2].
Marc-Olivier
MO
Ebert
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.
Patrick
P
Kiefer
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.
Dominik M
DM
Peter
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.
Julia A
JA
Vorholt
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.
Tobias J
TJ
Erb
D002384:Catalysis; D004798:Enzymes / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009249:NADP / Q000378:metabolism*
D004798:Enzymes; D009249:NADP