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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank


  1. 1

    Williamson, D.H., Lund, P. & Krebs, H.A. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J. 103, 514–527 (1967).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Veech, R.L., Eggleston, L.V. & Krebs, H.A. The redox state of free nicotinamide-adenine dinucleotide phosphate in the cytoplasm of rat liver. Biochem. J. 115, 609–619 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Sies, H. Metabolic compartmentation (Academic Press, 1982).

  4. 4

    Klingenberg, M. & Buecher, T. Biological oxidations. Annu. Rev. Biochem. 29, 669–708 (1960).

    Article  CAS  Google Scholar 

  5. 5

    Pollak, N., Dölle, C. & Ziegler, M. The power to reduce: pyridine nucleotides—small molecules with a multitude of functions. Biochem. J. 402, 205–218 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    MacDonald, M.J. Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets: further implication of cytosolic NADPH in insulin secretion. J. Biol. Chem. 270, 20051–20058 (1995).

    CAS  PubMed  Google Scholar 

  7. 7

    Ronnebaum, S.M. et al. A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion. J. Biol. Chem. 281, 30593–30602 (2006).

    Article  CAS  Google Scholar 

  8. 8

    Freeman, H.C., Hugill, A., Dear, N.T., Ashcroft, F.M. & Cox, R.D. Deletion of nicotinamide nucleotide transhydrogenase: a new quantitive trait locus accounting for glucose intolerance in C57BL/6J mice. Diabetes 55, 2153–2156 (2006).

    Article  CAS  Google Scholar 

  9. 9

    Pandolfi, P.P. et al. Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress. EMBO J. 14, 5209–5215 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Ducker, G.S. et al. Reversal of cytosolic one-carbon flux compensates for loss of the mitochondrial folate pathway. Cell Metab. 23, 1140–1153 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Chen, D. et al. Tissue-specific regulation of SIRT1 by calorie restriction. Genes Dev. 22, 1753–1757 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Sahlin, K., Katz, A. & Henriksson, J. Redox state and lactate accumulation in human skeletal muscle during dynamic exercise. Biochem. J. 245, 551–556 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    O'Neill, J.S. & Reddy, A.B. Circadian clocks in human red blood cells. Nature 469, 498–503 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Schwartz, J.P., Passonneau, J.V., Johnson, G.S. & Pastan, I. The effect of growth conditions on NAD+ and NADH concentrations and the NAD+:NADH ratio in normal and transformed fibroblasts. J. Biol. Chem. 249, 4138–4143 (1974).

    CAS  PubMed  Google Scholar 

  15. 15

    Braidy, N. et al. Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats. PLoS One 6, e19194 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Zhu, X.H., Lu, M., Lee, B.Y., Ugurbil, K. & Chen, W. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences. Proc. Natl. Acad. Sci. USA 112, 2876–2881 (2015).

    Article  CAS  Google Scholar 

  17. 17

    Verdin, E. NAD+ in aging, metabolism, and neurodegeneration. Science 350, 1208–1213 (2015).

    Article  CAS  Google Scholar 

  18. 18

    Zhao, Y. et al. SoNar, a highly responsive NAD+/NADH sensor, allows high-throughput metabolic screening of anti-tumor agents. Cell Metab. 21, 777–789 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Hung, Y.P., Albeck, J.G., Tantama, M. & Yellen, G. Imaging cytosolic NADH-NAD+ redox state with a genetically encoded fluorescent biosensor. Cell Metab. 14, 545–554 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Cameron, W.D. et al. Apollo-NADP+: a spectrally tunable family of genetically encoded sensors for NADP+. Nat. Methods 13, 352–358 (2016).

    Article  CAS  Google Scholar 

  21. 21

    Bilan, D.S. & Belousov, V.V. Genetically encoded probes for NAD+/NADH monitoring. Free Radic. Biol. Med. 100, 32–42 (2016).

    Article  CAS  Google Scholar 

  22. 22

    Fan, J. et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510, 298–302 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Liu, L. et al. Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage. Nat. Chem. Biol. 12, 345–352 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Lewis, C.A. et al. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol. Cell 55, 253–263 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Jiang, L. et al. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature 532, 255–258 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Titov, D.V. et al. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio. Science 352, 231–235 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Cahn, J.K. et al. A general tool for engineering the NAD/NADP cofactor preference of oxidoreductases. ACS Synth. Biol. 6, 326–333 (2017).

    Article  CAS  Google Scholar 

  28. 28

    Brown, D.M., Upcroft, J.A. & Upcroft, P. A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. Eur. J. Biochem. 241, 155–161 (1996).

    Article  CAS  Google Scholar 

  29. 29

    Lountos, G.T. et al. The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme. Biochemistry 45, 9648–9659 (2006).

    Article  CAS  Google Scholar 

  30. 30

    Scrutton, N.S., Berry, A. & Perham, R.N. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343, 38–43 (1990).

    Article  CAS  Google Scholar 

  31. 31

    Hanukoglu, I. & Gutfinger, T. cDNA sequence of adrenodoxin reductase: identification of NADP-binding sites in oxidoreductases. Eur. J. Biochem. 180, 479–484 (1989).

    Article  CAS  Google Scholar 

  32. 32

    Wallen, J.R., Paige, C., Mallett, T.C., Karplus, P.A. & Claiborne, A. Pyridine nucleotide complexes with Bacillus anthracis coenzyme A-disulfide reductase: a structural analysis of dual NAD(P)H specificity. Biochemistry 47, 5182–5193 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Krauth-Siegel, R.L., Arscott, L.D., Schönleben-Janas, A., Schirmer, R.H. & Williams, C.H. Jr. Role of active site tyrosine residues in catalysis by human glutathione reductase. Biochemistry 37, 13968–13977 (1998).

    Article  CAS  Google Scholar 

  34. 34

    Berry, A., Scrutton, N.S. & Perham, R.N. Switching kinetic mechanism and putative proton donor by directed mutagenesis of glutathione reductase. Biochemistry 28, 1264–1269 (1989).

    Article  CAS  Google Scholar 

  35. 35

    Tischler, M.E., Friedrichs, D., Coll, K. & Williamson, J.R. Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats. Arch. Biochem. Biophys. 184, 222–236 (1977).

    Article  CAS  Google Scholar 

  36. 36

    Eng, J., Lynch, R.M. & Balaban, R.S. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes. Biophys. J. 55, 621–630 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Morais, R., Gregoire, M., Jeannotte, L. & Gravel, D. Chick embryo cells rendered respiration-deficient by chloramphenicol and ethidium bromide are auxotrophic for pyrimidines. Biochem. Biophys. Res. Commun. 94, 71–77 (1980).

    Article  CAS  Google Scholar 

  38. 38

    King, M.P. & Attardi, G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246, 500–503 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Perham, R.N., Scrutton, N.S. & Berry, A. New enzymes for old: redesigning the coenzyme and substrate specificities of glutathione reductase. BioEssays 13, 515–525 (1991).

    Article  CAS  Google Scholar 

  40. 40

    Petschacher, B. et al. Cofactor specificity engineering of Streptococcus mutans NADH oxidase 2 for NAD(P)+ regeneration in biocatalytic oxidations. Comput. Struct. Biotechnol. J. 9, e201402005 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Rosell, A. et al. Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase. J. Biol. Chem. 278, 40573–40580 (2003).

    Article  CAS  Google Scholar 

  42. 42

    Chen, R., Greer, A. & Dean, A.M. Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase. Proc. Natl. Acad. Sci. USA 93, 12171–12176 (1996).

    Article  CAS  Google Scholar 

  43. 43

    Chen, R., Greer, A. & Dean, A.M. A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity. Proc. Natl. Acad. Sci. USA 92, 11666–11670 (1995).

    Article  CAS  Google Scholar 

  44. 44

    Wallen, J.R. et al. Structural analysis of Streptococcus pyogenes NADH oxidase: conformational dynamics involved in formation of the C(4a)-peroxyflavin intermediate. Biochemistry 54, 6815–6829 (2015).

    Article  CAS  Google Scholar 

  45. 45

    Pai, E.F. & Schulz, G.E. The catalytic mechanism of glutathione reductase as derived from X-ray diffraction analyses of reaction intermediates. J. Biol. Chem. 258, 1752–1757 (1983).

    CAS  PubMed  Google Scholar 

  46. 46

    Karplus, P.A. & Schulz, G.E. Substrate binding and catalysis by glutathione reductase as derived from refined enzyme: substrate crystal structures at 2 Å resolution. J. Mol. Biol. 210, 163–180 (1989).

    Article  CAS  Google Scholar 

  47. 47

    Krebs, H.A. & Veech, R.L. Equilibrium relations between pyridine nucleotides and adenine nucleotides and their roles in the regulation of metabolic processes. Adv. Enzyme Regul. 7, 397–413 (1969).

    Article  CAS  Google Scholar 

  48. 48

    Petcu, L.G. & Plaut, G.W. NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes. Biochem. J. 190, 581–592 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Burch, H.B., Bradley, M.E. & Lowry, O.H. The measurement of triphosphopyridine nucleotide and reduced triphosphopyridine nucleotide and the role of hemoglobin in producing erroneous triphosphopyridine nucleotide values. J. Biol. Chem. 242, 4546–4554 (1967).

    CAS  PubMed  Google Scholar 

  50. 50

    Tabor, H., Tabor, C.W. & Hafner, E.W. Convenient method for detecting 14CO2 in multiple samples: application to rapid screening for mutants. J. Bacteriol. 128, 485–486 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Carpenter, A.E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


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.

Author information




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.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–6 (PDF 2213 kb)

Supplementary Data Set 1

GC–MS data (XLSX 74 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading