Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Abstract

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423OLys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme. Milk xanthine oxidase is an archetypal enzyme, which was originally described as aldehyde oxidase in 1902 (1) and has since served as a benchmark for the whole class of complex metalloflavoproteins (2). Xanthine oxidoreductase enzymes have been isolated from a wide range of organisms, from bacteria to man, and catalyze the hydroxylation of a wide variety of purine, pyrimidine, pterin, and aldehyde substrates. All of these proteins have similar molecular weights and composition of redox centers (3, 4). The mammalian enzymes, which catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) and exist mostly as such in the cell but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. XDH shows a preference for NAD ϩ reduction at the flavin adenine dinucleotide (FAD) reaction site, whereas XO fails to react with NAD ϩ and exclusively uses dioxygen as its substrate, leading to the formation of superoxide anion and hydrogen peroxide (3). The enzyme is a target of drugs against gout and hyperuricemia (5), and the conversion of XDH to XO is of major interest as it has been implicated in diseases characterized by oxygen-radical-induced tissue damage, such as postischemic reperfusion injury (6). Recent work suggests that XO also might be associated with blood pressure regulation (7).The active form of the enzyme is that of a homodimer of molecular mass 290 kDa, with each of the monomers acting independently in catalysis. Each subunit contains one molybdopterin cofactor, two spectroscopically distinct [2Fe-2S] centers, and one FAD cofactor. The oxidation of xanthine takes place at the molybdopterin center (Mo-pt) and the electrons thus introduced are rapidly distributed to the other c...

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

“…The molybdenum atom is present in a distorted square pyramidal geometry, with one apical =O group and with the four equatorial positions occupied by two sulfur atoms of the cis-dithiolene (-S-C=C-S-) group of the pterin cofactor molecule, one essential =S group and one labile -OH/-OH 2 group [13]. Physiologically, XO is a key enzyme in purine catabolism, where it catalyses the hydroxylation of both hypoxanthine and xanthine to the terminal metabolite urate.…”

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

“…XD can, however, be readily converted to a ''strict'' oxidase form (named XO), either reversibly, through oxidation of the cysteine residues 535 and 992, or irreversibly, by proteolysis [5,6]. The cysteine oxidation (or proteolysis) causes a conformational change in the vicinity of the FAD, the site at which O 2 and NAD + react, increasing the midpoint potential of the flavin moiety and blocking the access of NAD + to FAD, but without disturbing the interactions between O 2 and FAD [7].…”

“…The reaction catalysed by XOR can be separated into a reductive half-reaction and an oxidative half-reaction. The reducing substrates are oxidised at the molybdenum site, and the reduction mechanism is thought to be the same for both XD and XO [1,7]. The electrons thus transferred from the substrate to the enzyme are rapidly distributed throughout the other centres, by intramolecular electron transfer, according to their redox potentials.…”

Regulation and Biochemistry of Mouse Molybdo-flavoenzymes

“…However, these differences do not significantly affect the catalytic activity of the DBA/2-derived oxidase, as transient transfection of the corresponding cDNA in a suitable host cell line results in expression of AOH1 enzymatic activity. Interestingly, molecular modeling of AOH1 against the known XOR crystal structure (44) indicates that the amino acid differences observed fall within domains that are predicted to have minor (if any) effects on the overall structure of the substrate binding domain. 4 Although we cannot formally exclude post-transcriptional events, the DBA/2 deficit of AOH1 is likely to be the consequence of a deficient transcription of the corresponding gene that leads to a dramatic decrease in the steady-state levels of the mature mRNA.…”