Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Jan;3(1):51–57. doi: 10.1002/pro.5560030107

A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase.

S Chen 1, P S Deng 1, J M Bailey 1, K M Swiderek 1
PMCID: PMC2142469  PMID: 7511454

Abstract

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bailey J. M., Nikfarjam F., Shenoy N. R., Shively J. E. Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate. Protein Sci. 1992 Dec;1(12):1622–1633. doi: 10.1002/pro.5560011210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayney R. M., Rodkey J. A., Bennett C. D., Lu A. Y., Pickett C. B. Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein. J Biol Chem. 1987 Jan 15;262(2):572–575. [PubMed] [Google Scholar]
  3. Benson A. M., Hunkeler M. J., Talalay P. Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5216–5220. doi: 10.1073/pnas.77.9.5216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen S., Hwang J., Deng P. S. Inhibition of NAD(P)H:quinone acceptor oxidoreductase by flavones: a structure-activity study. Arch Biochem Biophys. 1993 Apr;302(1):72–77. doi: 10.1006/abbi.1993.1182. [DOI] [PubMed] [Google Scholar]
  5. Chen S., Liu X. F. Suggested mechanism for the modulation of the activity of NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by menadione: interpretation of the effect of menadione on 5'-[p-(Fluorosulfonyl)benzoyl]adenosine labeling of rat liver NAD(P)H:quinone acceptor oxidoreductase. Mol Pharmacol. 1992 Sep;42(3):545–548. [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. Davis M. T., Lee T. D. Analysis of peptide mixtures by capillary high performance liquid chromatography: a practical guide to small-scale separations. Protein Sci. 1992 Jul;1(7):935–944. doi: 10.1002/pro.5560010712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. ERNSTER L., DANIELSON L., LJUNGGREN M. DT diaphorase. I. Purification from the soluble fraction of rat-liver cytoplasm, and properties. Biochim Biophys Acta. 1962 Apr 9;58:171–188. doi: 10.1016/0006-3002(62)90997-6. [DOI] [PubMed] [Google Scholar]
  9. Forrest G. L., Qian J., Ma J. X., Kaplan W. D., Akman S., Doroshow J., Chen S. A. Rat liver NAD(P)H:quinone oxidoreductase: cDNA expression and site-directed mutagenesis. Biochem Biophys Res Commun. 1990 Jun 29;169(3):1087–1093. doi: 10.1016/0006-291x(90)92006-l. [DOI] [PubMed] [Google Scholar]
  10. Haniu M., Yuan H., Chen S. A., Iyanagi T., Lee T. D., Shively J. E. Structure-function relationship of NAD(P)H:quinone reductase: characterization of NH2-terminal blocking group and essential tyrosine and lysine residues. Biochemistry. 1988 Sep 6;27(18):6877–6883. doi: 10.1021/bi00418a033. [DOI] [PubMed] [Google Scholar]
  11. Hollander P. M., Ernster L. Studies on the reaction mechanism of DT diaphorase. Action of dead-end inhibitors and effects of phospholipids. Arch Biochem Biophys. 1975 Aug;169(2):560–567. doi: 10.1016/0003-9861(75)90200-3. [DOI] [PubMed] [Google Scholar]
  12. Hosoda S., Nakamura W., Hayashi K. Properties and reaction mechanism of DT diaphorase from rat liver. J Biol Chem. 1974 Oct 25;249(20):6416–6423. [PubMed] [Google Scholar]
  13. Iyanagi T., Yamazaki I. One-electron-transfer reactions in biochemical systems. V. Difference in the mechanism of quinone reduction by the NADH dehydrogenase and the NAD(P)H dehydrogenase (DT-diaphorase). Biochim Biophys Acta. 1970 Sep 1;216(2):282–294. doi: 10.1016/0005-2728(70)90220-3. [DOI] [PubMed] [Google Scholar]
  14. Jaiswal A. K., Burnett P., Adesnik M., McBride O. W. Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6. Biochemistry. 1990 Feb 20;29(7):1899–1906. doi: 10.1021/bi00459a034. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Liu X. F., Liu M. L., Iyanagi T., Legesse K., Lee T. D., Chen S. A. Inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by flavonoids isolated from the Chinese herb scutellariae radix (Huang Qin). Mol Pharmacol. 1990 Jun;37(6):911–915. [PubMed] [Google Scholar]
  17. Liu X. F., Yuan H., Haniu M., Iyanagi T., Shively J. E., Chen S. A. Reaction of rat liver DT-diaphorase (NAD(P)H:quinone acceptor reductase) with 5'-[p-(fluorosulfonyl)benzoyl]-adenosine. Mol Pharmacol. 1989 Jun;35(6):818–822. [PubMed] [Google Scholar]
  18. Ma Q., Cui K., Xiao F., Lu A. Y., Yang C. S. Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis. J Biol Chem. 1992 Nov 5;267(31):22298–22304. [PubMed] [Google Scholar]
  19. Prestera T., Prochaska H. J., Talalay P. Inhibition of NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue and related anthraquinone dyes: a structure-activity study. Biochemistry. 1992 Jan 28;31(3):824–833. doi: 10.1021/bi00118a027. [DOI] [PubMed] [Google Scholar]
  20. Prochaska H. J. Purification and crystallization of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue affinity chromatography: identification of a new and potent inhibitor. Arch Biochem Biophys. 1988 Dec;267(2):529–538. doi: 10.1016/0003-9861(88)90060-4. [DOI] [PubMed] [Google Scholar]
  21. Robertson J. A., Chen H. C., Nebert D. W. NAD(P)H:menadione oxidoreductase. Novel purification of enzyme cDNA and complete amino acid sequence, and gene regulation. J Biol Chem. 1986 Nov 25;261(33):15794–15799. [PubMed] [Google Scholar]
  22. Ronk M., Shively J. E., Shute E. A., Blake R. C., 2nd Amino acid sequence of the blue copper protein rusticyanin from Thiobacillus ferrooxidans. Biochemistry. 1991 Oct 1;30(39):9435–9442. doi: 10.1021/bi00103a007. [DOI] [PubMed] [Google Scholar]
  23. Siegel D., Gibson N. W., Preusch P. C., Ross D. Metabolism of diaziquone by NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase): role in diaziquone-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 1990 Nov 15;50(22):7293–7300. [PubMed] [Google Scholar]
  24. Siegel D., Gibson N. W., Preusch P. C., Ross D. Metabolism of mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 1990 Dec 1;50(23):7483–7489. [PubMed] [Google Scholar]
  25. Skelly J. V., Suter D. A., Knox R. J., Garman E., Stuart D. I., Sanderson M. R., Roberts J. J., Neidle S. Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line. J Mol Biol. 1989 Feb 5;205(3):623–624. doi: 10.1016/0022-2836(89)90233-7. [DOI] [PubMed] [Google Scholar]
  26. Wallin R., Gebhardt O., Prydz H. NAD(P)H dehydrogenase and its role in the vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone)-dependent carboxylation reaction. Biochem J. 1978 Jan 1;169(1):95–101. doi: 10.1042/bj1690095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Walton M. I., Smith P. J., Workman P. The role of NAD(P)H: quinone reductase (EC 1.6.99.2, DT-diaphorase) in the reductive bioactivation of the novel indoloquinone antitumor agent EO9. Cancer Commun. 1991 Jul;3(7):199–206. doi: 10.3727/095535491820873164. [DOI] [PubMed] [Google Scholar]
  28. Ysern X., Prochaska H. J. X-ray diffraction analyses of crystals of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase containing cibacron blue. J Biol Chem. 1989 May 15;264(14):7765–7767. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES