Catalytic Properties of NAD(P)H:Quinone Oxidoreductase-2 (NQO2), a Dihydronicotinamide Riboside Dependent Oxidoreductase

doi:10.1006/abbi.1997.0344

Archives of Biochemistry and Biophysics

Volume 347, Issue 2, 15 November 1997, Pages 221-228

https://doi.org/10.1006/abbi.1997.0344 Get rights and content

Abstract

Human NAD(P)H:quinone acceptor oxidoreductase-2 (NQO2) has been prepared using anEscherichia coliexpression method. NQO2 is thought to be an isoform of DT-diaphorase (EC 1.6.99.2) [also referred to as NAD(P)H:quinone acceptor oxidoreductase] because there is a 49% identity between their amino acid sequences. The present investigation has revealed that like DT-diaphorase, NQO2 is a dimer enzyme with one FAD prosthetic group per subunit. Interestingly, NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. It catalyzes a two-electron reduction of quinones and oxidation–reduction dyes. One-electron acceptors, such as potassium ferricyanide, cannot be reduced by NQO2. This enzyme also catalyzes a four-electron reduction, using methyl red as the electron acceptor. The NRH-methyl red reductase activity of NQO2 is 11 times the NADH-methyl red reductase activity of DT-diaphorase. In addition, through a four-electron reduction reaction, NQO2 can catalyze nitroreduction of cytotoxic compound CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. NQO2 is 3000 times more effective than DT-diaphorase in the reduction of CB 1954. Therefore, NQO2 is a NRH-dependent oxidoreductase which catalyzes two- and four-electron reduction reactions. NQO2 is resistant to typical inhibitors of DT-diaphorase, such as dicumarol, Cibacron blue, and phenindone. Flavones are inhibitors of NQO2. However, structural requirements of flavones for the inhibition of NQO2 are different from those for DT-diaphorase. The most potent flavone inhibitor tested so far is quercetin (3,5,7,3′,4′-.6pentahydroxyflavone). It has been found that quercetin is a competitive inhibitor with respect to NRH (K_i= 21 nM). NQO2 is 43 amino acids shorter than DT-diaphorase, and it has been suggested that the carboxyl terminus of DT-diaphorase plays a role in substrate binding (S. Chenet al., Protein Sci.3, 51–57, 1994). In order to understand better the basis of catalytic differences between NQO2 and DT-diaphorase, a human NQO2 with 43 amino acids from the carboxyl terminus of human DT-diaphorase (i.e., hNQO2–hDT43) has been prepared. hNQO2–hDT43 still uses NRH as an electron donor. In addition, the chimeric enzyme is inhibited by quercetin but not dicumarol. These results suggest that additional region(s) in these enzymes is involved in differentiating NRH from NAD(P)H.

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NAD(P)H: Quinone Oxidoreductase 1 (NQO1) is an antioxidant enzyme that catalyzes the two-electron reduction of several different classes of quinone-like compounds (quinones, quinone imines, nitroaromatics, and azo dyes). One-electron reduction of quinone or quinone-like metabolites is considered to generate semiquinones to initiate redox cycling that is responsible for the generation of reactive oxygen species and oxidative stress and may contribute to the initiation of adverse drug reactions and adverse health effects. On the other hand, the two-electron reduction of quinoid compounds appears important for drug activation (bioreductive activation) via chemical rearrangement or autoxidation. Two-electron reduction decreases quinone levels and opportunities for the generation of reactive species that can deplete intracellular thiol pools. Also, studies have shown that induction or depletion (knockout) of NQO1 were associated with decreased or increased susceptibilities to oxidative stress, respectively. Moreover, another member of the quinone reductase family, NRH: Quinone Oxidoreductase 2 (NQO2), has a significant functional and structural similarity with NQO1. The activity of both antioxidant enzymes, NQO1 and NQO2, becomes critically important when other detoxification pathways are exhausted. Therefore, this article summarizes the interactions of NQO1 and NQO2 with different pharmacological agents, endogenous biochemicals, and environmental contaminants that would be useful in the development of therapeutic approaches to reduce the adverse drug reactions as well as protection against quinone-induced oxidative damage. Also, future directions and areas of further study for NQO1 and NQO2 are discussed.
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Interest in pharmacological agents capable of increasing cellular NAD⁺ concentrations has stimulated investigations of nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). NR and NMN require large dosages for effect. Herein, we describe synthesis of dihydronicotinamide riboside (NRH) and the discovery that NRH is a potent NAD⁺ concentration-enhancing agent, which acts within as little as 1 h after administration to mammalian cells to increase NAD⁺ concentrations by 2.5–10-fold over control values. Comparisons with NR and NMN show that in every instance, NRH provides greater NAD⁺ increases at equivalent concentrations. NRH also provides substantial NAD⁺ increases in tissues when administered by intraperitoneal injection to C57BL/6J mice. NRH substantially increases NAD⁺/NADH ratio in cultured cells and in liver and no induction of apoptotic markers or significant increases in lactate levels in cells. Cells treated with NRH are resistant to cell death caused by NAD⁺-depleting genotoxins such as hydrogen peroxide and methylmethane sulfonate. Studies to identify its biochemical mechanism of action showed that it does not inhibit NAD⁺ consumption, suggesting that it acts as a biochemical precursor to NAD⁺. Cell lysates possess an ATP-dependent kinase activity that efficiently converts NRH to the compound NMNH, but independent of Nrk1 or Nrk2. These studies identify a putative new metabolic pathway to NAD⁺ and a potent pharmacologic agent for NAD⁺ concentration enhancement in cells and tissues.
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Citation Excerpt :
Here we present an example of how a mechanistic understanding of the pH-dependence of the hydroquinone oxidation by oxygen, and its interaction with copper, could be employed to test hypotheses of biochemical relevance. In biological systems, the enzymes NQO1 and NQO2 (NAD(P)H:quinone oxidoreductase-2) catalyse the two-electron reduction of menadione (and other quinones) to the hydroquinone form (menadiol) [62]. Both enzymes are found in human tissues (e.g., brain, lung, liver, etc.) [63].
The oxidation of hydroquinones is of interest both due to the generation of reactive oxygen species (ROS) and to the implications to trace metal redox state. Menadione (MNQ), a typical toxicant quinone used extensively for studying the mechanisms underlying oxidative stress, is known to be an effective source of exogenous ROS. In this study, the kinetics and mechanism of the oxidation of menadiol (MNH₂Q, the reduced form of MNQ) in the absence and presence of copper (Cu) over the pH range 6.0–7.5 was examined. The autoxidation rate increased with increasing pH and concentration of O₂ and also slightly increased with increasing concentration of MNH₂Q and MNQ with Cu shown to play a significant role in catalysing the oxidation of MNH₂Q. A kinetic model showed that the mono-deprotonated menadiol, MNHQ⁻, accounted for the pH dependence of the autoxidation rate. In this proposed mechanism, both MNH₂Q and MNHQ⁻ species were oxidized quickly by Cu(II), generating menadione semiquinone (MNSQ^•−) and superoxide (O₂^•−) and the reduced form of Cu, Cu(I). Oxygen not only facilitated the catalytic role of Cu(II) by rapidly regenerating Cu(II) but also effectively removed MSNQ^•−, generating the important chain-propagating species O₂^•−. The model demonstrated that Cu(I) was a significant sink of O₂^•− resulting in the generation of H₂O₂ with subsequent generation of highly oxidative intermediates including Cu(III). These results provide considerable insight into the clinical significance of the biological activation and detoxification of MNQ with the kinetic model developed of use in identifying key processes in the generation of harmful oxidants in living systems.
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^☆: This research was supported by American Heart Association Grant 95007330 (S.C.), NIH Grants CA44735 (S.C.) and CA33572 (City of Hope Cancer Center), a Seed Grant from the City of Hope (S.C.), UK Cancer Research Campaign (R.K.), and NIH Grant ES07943 (A.K.J.).

^☆☆: K. Yagi, Ed.

²: To whom correspondence should be addressed. Fax: (818) 301-8186. E-mail: [email protected].

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Regular ArticleCatalytic Properties of NAD(P)H:Quinone Oxidoreductase-2 (NQO2), a Dihydronicotinamide Riboside Dependent Oxidoreductase☆,☆☆

Abstract

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Biochem. Pharmacol.

Arch. Biochem. Biophys.

Regular Article
Catalytic Properties of NAD(P)H:Quinone Oxidoreductase-2 (NQO2), a Dihydronicotinamide Riboside Dependent Oxidoreductase☆,☆☆