The Crystal Structure of NAD(P)H Quinone Oxidoreductase 1 in Complex with Its Potent Inhibitor Dicoumarol

Asher, Gad; Dym, Orly; Tsvetkov, Peter; Adler, Julia; Shaul, Yosef

doi:10.1021/bi0600087

Cited by 155 publications

(224 citation statements)

References 25 publications

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“…The peak at 450 nm suggests that the FAD is unreduced in the presence of dicumarol. This result corroborates very well the observations from the NQO1-dicumarol complex crystal structure analysis reported earlier [34].…”

Section: Discussionsupporting

confidence: 93%

“…The regeneration of FAD implies that E 2 -3,4-Q has reached the binding pocket and it did not alkylate the binding site, corroborating the interpretation of our ESI-MS results. Based on the crystal structure of the NQO1-dicumarol complex, the inhibitor is thought to occupy the position in the binding pocket that is meant for NADH [34]. The effect of the competitive inhibitor dicumarol on the reduction of FAD-NQO1 is depicted in Figure 4.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones

Gaikwad

Rogan

Cavalieri

2007

Free Radical Biology and Medicine

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Estrogen ortho-quinones have been implicated as ultimate carcinogenic metabolites of estrogens. The present conclusion that estrogen ortho-quinones are not substrates for NAD(P)H:quinone oxidoreductase (NQO1) stems from earlier reports. In this investigation, we were successful in circumventing the problem of nonenzymatic reduction of estrogen quinone by NAD(P)H, which led to the above conclusion, and for the first time show that NQO1 catalyzes the reduction of estrogen quinones. Mass spectrometric binding studies involving estradiol-3,4-quinone or menadione with NQO1 clearly support the formation of an enzyme-substrate physical complex. However, the NQO1 mass spectrum did not alter after addition of cholesterol, the control. Two different strategies were employed to ascertain the NQO1 activity in estrogen quinone reduction. First, the ping-pong mechanism of NQO1 catalysis was utilized to overcome the problem of nonenzymatic reduction of the substrate by NADH. Second, tetrahydrofolic acid, which has a lower reducing potential, was used as an alternate cofactor. Both of these methods confirmed the reduction of estradiol-3,4-quinone by NQO1, when assay mixtures were analyzed by UV or liquid chromatography-mass spectrometry. Furthermore, reduction of 9,10-phenanthrene quinone or menadione was observed using the reported assay conditions. Thus, clear evidence for the catalytic reduction of estrogen ortho-quinones by NQO1 has been obtained; its mechanism and implications are discussed.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones

Gaikwad

Rogan

Cavalieri

2007

Free Radical Biology and Medicine

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“…Studies of NQO1 structure and function have shown that the NAD(P)H and the quinone binding sites of enzymes have a significant overlap (25), and the enzymatic mechanism is a typical ping-pong reaction. In this study, two coumarin derivatives (warfarin and 6,7-dihydroxycoumarin) (26,27), dicoumarol (28), and the reactive dye Cibacron Blue (Reactive Blue-2 (RB-2)) (29,30), which compete with NAD(P)H for binding to NQO1, were tested to see whether these compounds inhibit Ndi1. The structures of these inhibitors are shown in Fig.…”

Section: Fad Contents and Nadh Oxidase Activities Of Mutant Enzymes-mmentioning

confidence: 99%

Reaction Mechanism of Single Subunit NADH-Ubiquinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

Yu¹,

Yamashita²,

Nakamaru‐Ogiso

et al. 2011

Journal of Biological Chemistry

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The flavoprotein rotenone-insensitive internal NADHubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin. Alternative NADH dehydrogenases (NDH-2)4 catalyze electron transfer from NADH to quinone without energy transduction. They are commonly found in the respiratory chain of bacteria, fungi, and plant mitochondria but not in mammalian mitochondria (1, 2). The NDH-2 enzyme is generally composed of a single polypeptide and can be classified into three groups: A, B, and C. Group A has two adenine dinucleotide (ADP)-binding motifs and is involved in the non-covalent binding of NAD(P)H and FAD. Group B has the same two ADP-binding motifs and an additional conserved EF-hand. Group C has only one conserved ADP-binding motif where flavin is covalently bound (2). The yeast Ndi1 enzyme belongs to group A (2) and catalyzes NADH oxidation on the matrix side of the mitochondrial inner membrane (3) like the energy-transducing NADH dehydrogenase complex I (4).The physiological electron acceptor of NDH-2 is quinone, which is broadly classified into two types: naphthoquinone (menaquinone) and benzoquinone (ubiquinone (UQ)). Ndi1 utilizes the latter as an electron acceptor (2). Both quinones have a hydrophilic head group and hydrophobic isoprenoid side chains and ensure electron transfer between several membrane-bound respiratory enzymes. Ndi1 does not react with oxygen in the presence of UQ. In the absence of quinone, NADH-reduced Ndi1 reacts with oxygen and produces H 2 O 2 , although intrinsic NADH oxidase activity is extremely low (about 30...

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“…Dicumarol에 의한 PMA 유도 MMP-9 발현 억제에 NQO1 관련성 조사 Dicumarol은 NQO1의 효소 활성 저해제롤 잘 알려져 있다 [4,14]. 따라서 dicumarol의 PMA 유도 MMP-9 활성 억제에 NQO1의 관련성을 확인하기 위하여 NQO1의 siRNA 처리에 의한 knock-down 시 PMA 유도 MMP-9 발현 및 wound healing migration 및 invasion assay를 실시하였다.…”

Section: 신장암세포인 Caki 세포에서 Pma유도 Mmp-9 활성화에 Dicumarol의 효과 확인unclassified

“…또한 NQO1효소의 NAD(P) 결합을 경쟁적으로 작용하므로 NQO1 효소 저해제 작용을 한다 [4]. 최근 인간 혈관 평활근세포에서 TNF-α에 의한 MMP-9 발현 및 분비에 NQO1의 관련성을 NQO-1 siRNA 실험을 통하여 제시하였다 [11].…”

Section: 신장암세포인 Caki 세포에서 Pma유도 Mmp-9 활성화에 Dicumarol의 효과 확인unclassified

Dicumarol Inhibits PMA-Induced MMP-9 Expression through NQO1-independent manner in Human Renal Carcinoma Caki Cells

Park¹,

Kwon²

2016

Journal of Life Science

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Dicumarol is a coumarin derivative isolated from sweet clover (Melilotus alba), and has anti-coagulant activity with the inhibitory activity of NAD(P)H quinone oxidoreductase1 (NQO1). NQO1 catalyzes the two-electron reduction of quinones to hydroquinones. Dicumarol competes with NAD(P)H for binding to NQO1, resulting in the inhibition of NQO1 enzymatic activity. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). However, the effects of dicumarol on metalloproteinase (MMP)-9 expression and activity are not investigated here. This study investigated whether dicumarol inhibits MMP-9 expression and activity in PMA-treated human renal carcinoma Caki cells. Dicumarol markedly inhibited the PMA-induced MMP-9 mRNA expression and MMP-9 activity. NF-κB and AP1 promoter activity, which is important in MMP-9 expression, also decreased in dicumarol-treated cells. Furthermore, dicumarol markedly suppressed the ability of PMA-mediated migration in Caki cells. When the relevance of NQO1 in the dicumarol-mediated inhibitory effect on PMA-induced MMP9 activity was elucidated, knock-down of NQO1 with siRNA was found to have no effect on PMA-induced MMP9 activity, suggesting that the stimulating effect of dicumarol on PMA-induced MMP9 activity is independent of NQO1 activity. Taken together, the present studies suggested that dicumarol may inhibit PMA-induced migration via down-regulation of MMP-9 expression and activity.

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The Crystal Structure of NAD(P)H Quinone Oxidoreductase 1 in Complex with Its Potent Inhibitor Dicoumarol

Cited by 155 publications

References 25 publications

Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones

Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones

Reaction Mechanism of Single Subunit NADH-Ubiquinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

Dicumarol Inhibits PMA-Induced MMP-9 Expression through NQO1-independent manner in Human Renal Carcinoma Caki Cells

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