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

Nature Chemical Biology volume 10, pages 50–55 (2014)Cite this article

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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.

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Figure 1: Spectrophotometric assay of Ccr activity identifies a transient intermediate during the reduction reaction.
Figure 2: NMR characterization of the transient intermediate.
Figure 3: Purification and characterization of the ene adduct.
Figure 4: Possible reaction mechanisms for the formation of the ene adduct.

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Acknowledgements

This work was supported by a Schweizerische Nationalfonds–Ambizione grant (PZ00P3_136828/1) and an ETH fellowship to T.J.E. We thank B.M. Jaun for valuable discussions and critical inspection of NMR data, A.J. Rosenthal for suggestions during the early phase of this work and S. Weidenweber and U. Ermler for discussions about a structural understanding of Ccr. We acknowledge K. Hiltunen, J. Torkko and A. Kastaniotis (University of Oulu, Finland) for providing the etr1p gene, G. Sturm (Albert-Ludwigs-University of Freiburg, Germany) for providing plasmid pGS5, A. Essig, M. Künzler and M. Aebi (ETH Zurich, Switzerland) for help with the Pichia pastoris expression system, R. Kissner and C. Molina (ETH Zurich, Switzerland) for stopped-flow experiments, S. Weidenweber and C. Vogel for the initial cloning of the Ccr and Etr1p constructs, respectively, and National BioResource Project (NBRP)-E. coli at the National Institute of Genetics (NIG) for providing E. coli strain JW3222.

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  1. Raoul G Rosenthal and Marc-Olivier Ebert: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland

    Raoul G Rosenthal, Patrick Kiefer, Dominik M Peter, Julia A Vorholt & Tobias J Erb

  2. Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland

    Marc-Olivier Ebert

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  1. Raoul G Rosenthal
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Contributions

R.G.R. and T.J.E. conceived and designed all experiments, with the exception of the NMR experiments, which were designed together with M.-O.E., and the MS analyses, which were designed together with P.K. and J.A.V. NMR experiments were performed by R.G.R. and M.-O.E. MS experiments were performed by R.G.R. and P.K. Enzyme kinetic assays and stopped-flow measurements, as well as purification of the intermediate, were performed by R.G.R. and D.M.P. R.G.R. and T.J.E. wrote the paper.

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Correspondence to Tobias J Erb.

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Rosenthal, R., Ebert, MO., Kiefer, P. et al. Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes. Nat Chem Biol 10, 50–55 (2014). https://doi.org/10.1038/nchembio.1385

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