Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase.
Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 Angstroms resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.
Asai
Ryosuke
R
Department of Biochemistry and Department of Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Tokyo, Japan.
Nishino
Tomoko
T
Matsumura
Tomohiro
T
Okamoto
Ken
K
Igarashi
Kiyohiko
K
Pai
Emil F
EF
Nishino
Takeshi
T
D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D004229:Dithiothreitol / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D017382:Reactive Oxygen Species; D018411:Spodoptera / Q000166:cytology; D013481:Superoxides / Q000378:metabolism; D014968:Xanthine Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D014969:Xanthine Oxidase / Q000737:chemistry* / Q000378:metabolism
D017382:Reactive Oxygen Species; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D004229:Dithiothreitol