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A genetically encoded tool for manipulation of NADP+/NADPH in living cells

Nature Chemical Biology volume 13, pages 1088–1095 (2017)Cite this article

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Abstract

The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

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Figure 1: Engineering TPNOX, a water-forming NADPH oxidase.
Figure 2: Catalytic activity of recombinant TPNOX.
Figure 3: Structural determinants of NAD(P)H substrate selectivity in LbNOX and TPNOX.
Figure 4: Expression and activity of TPNOX in human cells.
Figure 5: Metabolic consequences of compartment-specific perturbation of NADH or NADPH.

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Acknowledgements

We thank D. Trono (Swiss Federal Institute of Technology Lausanne) for providing reagents. This work was supported by grants R01GM099683 and R35GM122455 from the National Institutes of Health and by the Harvard Ludwig Center. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. Metabolomics analysis was performed at the Metabolomics Core Facility at the University of Utah, which is supported by 1 S10 OD016232-01, 1 S10 OD021505-01 and 1 U54 DK110858-01. V.K.M. is supported as an Investigator of the Howard Hughes Medical Institute from the National Institutes of Health.

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  1. Valentin Cracan and Denis V Titov: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Valentin Cracan, Denis V Titov, Hongying Shen, Zenon Grabarek & Vamsi K Mootha

  2. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Valentin Cracan, Denis V Titov, Hongying Shen & Vamsi K Mootha

  3. Broad Institute, Cambridge, Massachusetts, USA

    Valentin Cracan, Denis V Titov, Hongying Shen & Vamsi K Mootha

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Contributions

V.C., D.V.T., Z.G. and V.K.M. designed the study and interpreted data. V.C. and Z.G. performed the biochemical and structure-related experiments. D.V.T., V.C. and H.S. performed cell-based experiments. V.C., D.V.T., Z.G. and V.K.M. wrote the manuscript.

Corresponding authors

Correspondence to Zenon Grabarek or Vamsi K Mootha.

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Competing interests

V.C., D.T., Z.G. and V.K.M. are listed as inventors on a patent application filed by Massachusetts General Hospital on the LbNOX and TPNOX technology.

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Cracan, V., Titov, D., Shen, H. et al. A genetically encoded tool for manipulation of NADP+/NADPH in living cells. Nat Chem Biol 13, 1088–1095 (2017). https://doi.org/10.1038/nchembio.2454

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