Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a potent producer of superoxide anions via its NADH oxidase activity
- 596 Downloads
Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD+ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD+ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD+ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD+-dependent xanthine oxidation indicates that both NAD+ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.
KeywordsXanthine dehydrogenase AtXDH1 NADH oxidation Superoxide Superoxide dismutase Pichia pastoris
We are grateful to Prof. Dr. Guenter Schwarz and Dr. Jose Santamaria Araujo (University of Cologne) for their support in protein purification, and we thank Ute Nielaender and Victoria Michael (TU Braunschweig) for excellent technical assistance.
- Amaya Y, Yamazaki K, Sato M, Noda K, Nishino T, Nishino T (1990) Proteolytic conversion of xanthine dehydrogenase from the NAD-dependent type to the O2-dependent type. Amino acid sequence of rat liver xanthine dehydrogenase and identification of the cleavage sites of the enzyme protein during irreversible conversion by trypsin. J Biol Chem 265:14170–14175PubMedGoogle Scholar
- Avis PG, Bergel F, Bray RC (1955) Cellular constituents. The chemistry of xanthine oxidase. Part I. The preparation of a crystalline xanthine oxidase from cow’s milk. J Chem Soc 1100-–1105Google Scholar
- Avis PG, Bergel F, Bray RC (1956) Cellular constituents. The chemistry of xanthine oxidase. Part III. Estimations of cofactors and the catalytic functions of enzyme fractions of cow’s milk. J Chem Soc 1219–1225Google Scholar
- Boland MJ (1981) NAD+: xanthine dehydrogenase from nodules of navy beans: partial purification and properties. Biochem Int 2:567–574Google Scholar
- Corpas FJ, Gómez M, Hernández JA, Del Río LA (1993) Metabolism of activated oxygen in peroxisomes from two Pisum sativum L. cultivars with different sensitivity to sodium chloride. J Plant Physiol 141:160–165Google Scholar
- Corpas FJ, De La Colina C, Sanchez-Rasero F, Del Rio LA (1997) A role for leaf peroxisomes in the catabolism of purines. J Plant Physiol 151:246–250Google Scholar
- Massey V, Edmondson D (1970) On the mechanism of inactivation of xanthine oxidase by cyanide. J Biol Chem 244:1682–1691Google Scholar
- Montalbini P (2000) Xanthine dehydrogenase from leaves of leguminous plants: purification, characterization and properties of the enzyme. J Plant Phys 156:3–16Google Scholar
- Nishino T, Nishino T (1989) The nicotinamide adenine dinucleotide-binding site of chicken liver xanthine dehydrogenase. Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide. J Biol Chem 264:5468–5473PubMedGoogle Scholar
- Nishino T, Okamoto K, Kawaguchi Y, Hori H, Matsumura T, Eger BT, Pai EF, Nishino T (2005) Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant. J Biol Chem 280:24888–24894CrossRefPubMedGoogle Scholar