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

Rosenthal, R. G.; Ebert, M. O.; Kiefer, P.; Peter, D. M.; Vorholt, J. A.; Erb, T. J.

doi:10.1038/nchembio.1385

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

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https://www.ncbi.nlm.nih.gov/pubmed/24240506
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Rosenthal, R. G., Ebert, M. O., Kiefer, P., Peter, D. M., Vorholt, J. A., & Erb, T. J. (2013). Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes. Nat Chem Biol, 10(1), 50-5. doi:10.1038/nchembio.1385.

Cite as: https://hdl.handle.net/21.11116/0000-000A-CB0C-5

Abstract

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.