Unexpected genetic and structural relationships of a long-forgotten flavoenzyme to NAD(P)H:quinone reductase (DT-diaphorase)

A mammalian cytosolic FAD-dependent enzyme that catalyzes the reduction of quinones by N-ribosyl- and N-alkyldihydronicotinamides, but not by NADH, NADPH, or NMNH (reduced nicotinamide mononucleotide), was isolated from bovine kidney more than 30 years ago [S. Liao, J. T. Dulaney and H. G. Williams-Ashman (1962) J. Biol. Chem. 237, 2981–2987]. This enzyme is designated here as quinone reductase type 2 (QR(2)). Bovine QR(2) is a homodimer that migrates on SDS/PAGE at ≈22 kDa. Three tryptic peptides of bovine QR(2) (representing 39 amino acids) showed 43% identity to human NAD(P)H:quinone reductase (DT-diaphorase; EC 1.6.99.2), here designated QR(1) and 82% identity to a related human cDNA clone [called hNQO(2) by A. K. Jaiswal, P. Burnett, M. Adesnik and O. W. McBride (1990) Biochemistry 29, 1899–1906], and designated here as hQR(2). The protein encoded by the latter cDNA did not show QR activity when tested with conventional nicotinamide nucleotides. The unexpected high homology between the old flavoenzyme and hQR(2) prompted us to clone and overexpress hQR(2). The properties of hQR(2) were identical to those of the flavoenzyme described by S. Liao and H. G. Williams-Ashman, thus establishing their genetic identity. Recombinant human QR(2): (i) reacts with N-ribosyl- and N-alkyldihydronicotinamides, but not with NADH, NADPH, or NMNH; (ii) is very weakly inhibited by dicumarol or Cibacron blue; (iii) is very potently inhibited by benzo[a]pyrene. The x-ray crystal structure of rat QR(1) shows that the 43 amino acid C-terminal tail of QR(1) provides the binding site for the hydrophilic portions of NADH and NADPH. In the absence of this binding site in QR(2), the enzyme retains the essential catalytic machinery, including affinity for FAD, but cannot bind phosphorylated hydride donors.