Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: Species comparison and structural changes with substrate binding and release

Abstract

NAD(P)H͞quinone acceptor oxidoreductase (QR1, NQO1, formerly DT-diaphorase; EC 1.6.99.2) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. In this paper we report the apoenzyme structures of human (at 1.7-Å resolution) and mouse (2.8 Å) QR1 and the complex of the human enzyme with the substrate duroquinone (2.5 Å) (2,3,5,6-tetramethyl-p-benzoquinone). In addition to providing a description and rationale of the structural and catalytic differences among several species, these structures reveal the changes that accompany substrate or cofactor (NAD) binding and release. Tyrosine-128 and the loop spanning residues 232-236 close the binding site, partially occupying the space left vacant by the departing molecule (substrate or cofactor). These changes highlight the exquisite control of access to the catalytic site that is required by the ping-pong mechanism in which, after reducing the flavin, NAD(P) ؉ leaves the catalytic site and allows substrate to bind at the vacated position. In the human QR1-duroquinone structure one ring carbon is significantly closer to the flavin N5, suggesting a direct hydride transfer to this atom.DT diaphorase ͉ cancer ͉ chemoprotection ͉ chemotherapy N AD(P)H:Quinone acceptor oxidoreductase type 1 (QR1, NQO1; EC 1.6.99.2) is a flavoenzyme (homodimer of 273 residues, one FAD per monomer) that catalyzes the obligatory two-electron reduction of quinones to hydroquinones (1-3). This reaction prevents the one-electron reduction of quinones by cytochrome P450 reductase and other flavoproteins, resulting in oxidative cycling of deleterious radical species. The enzyme is inducible by a wide variety of Michael reaction acceptors and other electrophiles (4, 5). In addition to its possible role in the detoxification of dietary quinones, the enzyme has been shown to catalyze the reductive activation of quinolic chemotherapeutic compounds such as mitomycins (6), anthracyclines, and aziridinyl-benzoquinones. Overexpression of QR1 in many tumors, including those of lung, colon, liver, and breast, makes this enzyme an ideal target for the development of additional activatable cytotoxic compounds.We previously reported the crystal structures of rat liver quinone reductase (rQR1) in complex with NADP ϩ and the ternary complex with the inhibitor Cibacron Blue (CB) and the substrate duroquinone (2,3,5,6-tetramethyl-1,2-benzoquinone; DQ) (7). These structures have revealed insight into many biochemical and physiological properties of QRs, as well as the mechanism of quinone reduction. These structures have provided a wealth of information, including the overall fold, the nature of the dimer, the interactions of the FAD cofactor, and the possibility of hydride transfer between the NADH and FAD cofactors and from FADH 2 to the quinone substrate. Nonetheless many important aspects of QR1 function remain elusive. This is caused in part by the lack of structural information about the apoenzyme (apo) containing only the FAD prosthetic group (apo QR1) or of QR1...

A Novel Mechanism for Azoreduction

“…The enzymes showing azoreductase activity, for which there are structural data, all share a common core flavodoxin-like fold, 12,13,[40][41][42] although the sequence identity is low (Fig. 1).…”

Flavin-dependent quinone reductases

“…This of course may explain the incapacity of NQO2 to use classical hydride donors such as NAD(P)H. In NQO1, aromatic stacking of the nicotinamide ring and the flavin isoalloxazine ring provides additional stabilization of NAD(P)H. This kind of interaction may also be responsible for the binding and stabilization of nonphosphorylated electron-donating cosubstrates in the NQO2 active site, as this enzyme is capable of using both polar and non-polar N-substituted dihydronicotinamide substrates [9]. Two NQO1 structures (human and rat) show the catalytic pocket occupied by duroquinone (2,3,4,6-tetramethyl-1,4-benzoquinone), a small quinone sub- [7,34]. Duroquinone occupies nearly the same position vacated by the nicotinamide ring of NADP + .…”

FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1

“…enzymes such as MdaB [1][2][3]. The family members are typically dimeric 44 and have an FAD cofactor bound to each subunit [4][5][6][7]. They catalyse the 45 reduction of quinones (and related compounds) through a substituted 46 enzyme ("ping-pong") mechanism in which NAD(P)H (or NRH in the 47 case of NQO2) enters the active site, reduces the FAD and exits as the 48 oxidised form.…”