Abstract
Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.
Highlights
Xanthine oxidoreductase (XOR)1 is a ubiquitous metalloflavo enzyme with a central role in purine catabolism where it catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid
The present study has demonstrated that A. thaliana possesses duplicated XOR genes that are arranged in tandem orientation in the genome
Whereas xanthine dehydrogenase (XDH) is the predominant enzymatic form in normal tissues and is involved in purine catabolism xanthine oxidase (XO) dominates in milk and in tissues subjected to injury where the production of reactive oxygen species is required
Summary
Plant XDH is supposed to play a role in important cellular processes: (i) plant-pathogen interactions between phytopathogenic fungi, legumes, and cereals [21, 22]; (ii) cell death associated with hypersensitive response [23, 24]; and (iii) natural senescence [25]. As all these processes require the formation of reactive oxygen species XDH was supposed to be able to produce superoxide anions and/or hydrogen peroxide [25]. Pichia pastoris with subsequent purification and characterization of the AtXDH1 protein
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