Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase

Abstract

In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD ؉ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH͞XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and sitedirected mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423-Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.X anthine oxidoreductase (XOR) is a homodimer of molecular weight 290,000, and each subunit of the enzyme contains one molybdo-pterin (Mo-pt) cofactor, two distinct [2Fe-2S] centers, and one flavin adenine dinucleotide (FAD) cofactor (1, 2). The mammalian XORs catalyze the hydroxylation of hypoxanthine or xanthine at the Mo center, and reducing equivalents thus introduced into the enzymes are transferred via two [2Fe-2S] centers to FAD, where the reduction of NAD ϩ or molecular oxygen occurs (3). These enzymes are synthesized as the dehydrogenase form [xanthine dehydrogenase (XDH)] but can be readily converted to the oxidase form [xanthine oxidase (XO)] reversibly by oxidation of sulfhydryl residues or irreversibly by proteolysis (1, 2, 4-6). XDH shows a preference for NAD ϩ reduction at the FAD reaction site (although it still displays considerable reactivity with oxygen), whereas XO fails to react with NAD ϩ and exclusively uses dioxygen as its substrate, leading to formation of superoxide anion and hydrogen peroxide (1, 7). Previous investigations have suggested that the XDH͞XO conversion is related to milk lipid secretion (8, 9) and is implicated in diseases characterized by oxygen radical-induced tissue damage such as postischemic reperfusion injury (10-13). Thus, the XDH͞XO transition has attracted much attention from both basic and clinical researchers, not only because of the mechanistic interest in the different reactivity of FAD toward NAD ϩ or oxygen subs...

Regulation and Biochemistry of Mouse Molybdo-flavoenzymes

“…Animals were treated according to the standards set by Italian Law. 2 All animal experiments were approved by the Internal Animal Care and Use Committee and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996 edition) and European Union directives and guidelines. 3 Western Blot and Zymogram Analyses-The anti-AOH1-, anti-AOX1-, anti-AOH2-specific antibodies have been described previously (12).…”

“…Xanthine oxidoreductase (XOR) 1 (2,3) and aldehyde oxidase (AOX1) (4,5) are the two most thoroughly studied members of the family. XOR is involved in the catabolism of purines, oxidizing hypoxanthine into xanthine and xanthine into uric acid (1,6,7).…”

The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition

“…XDH shows a preference for NAD ϩ as the oxidizing substrate (although it is also able to react with O 2 ), whereas XO is unable to react with NAD ϩ and uses dioxygen exclusively (1). X-ray crystal structures of both the XDH and XO forms of bovine XOR have recently been reported at 2.1-and 2.5-Å resolution (5), respectively, and a detailed comparison of the two forms and site-directed mutagenesis studies has elucidated the structural basis for the XDH to XO switch (6). Based on the results of extended x-ray absorption fine-structure studies (2), two thiolene sulfurs (OSO), one sulfo (AS), one oxo (AO), and one hydroxo (OH) group or water had previously been postulated as the ligands of the active-site molybdenum center ( Fig.…”

Mechanism of the Conversion of Xanthine Dehydrogenase to Xanthine Oxidase

“…The proteolytic cleavage site is also on the linker peptide. Based on detailed analyses of crystal structures of reversible XDH and proteolytic XO, as well as site-directed mutagenesis, Kuwabara et al (19) (15).…”