Tandem Orientation of Duplicated Xanthine Dehydrogenase Genes from Arabidopsis thaliana

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 desulfointo 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. 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 enzyme from higher eukaryotes is active as a homodimer composed of two identical subunits of 150 kDa, each being subdivided into three domains: a N-terminal domain of 20 kDa for binding of two iron-sulfur clusters of the [2Fe-2S] type, a 40-kDa domain harboring a FAD-binding site, and a C-terminal molybdenum cofactor (Moco)-binding domain of 85 kDa. XOR enzymes in mammals are present either as the predominantly existing xanthine dehydrogenase (XDH; EC 1.1.1.204) or as O 2 -dependent xanthine oxidase (XO; EC 1.1.3.22). Whereas XDH possesses high reactivity toward NAD ϩ and low reactivity toward O 2 as electron acceptor, XO reacts in a strictly O 2 -dependent manner with negligible reactivity toward NAD ϩ . Both forms can be interconverted reversibly by oxidation of cysteine residues (1), whereas the conversion of XDH into XO by limited proteolysis is irreversible (2). In contrast to mammalian XOR the avian enzyme is exclusively present in the dehydrogenase form (3). The ability of mammalian XO to produce superoxide and hydrogen peroxide by reducing molecular oxygen (4) led to the suggestion that it might play an important role i...

Glutaredoxin GRXS17 Associates with the Cytosolic Iron-Sulfur Cluster Assembly Pathway

“…XDH1 and XDH2. Although both genes are located close to each other on the same chromosome, XDH2 is expressed constitutively at basal levels, whereas XDH1 is differentially expressed upon several developmental and environmental stimuli (Hesberg et al, 2004). Plants deficient in XDH1 are affected in growth and development.…”

“…XDH1 activity measurements in plant crude extracts were performed as described in Hesberg et al (2004). Briefly, total protein extract was obtained from 10-d-old Arabidopsis seedlings.…”

Biosíntesis, señalización y modo de acción del óxido nítrico en Arabidopsis thaliana

“…Tras seleccionar individuos con la inserción de T-DNA en homocigosis por PCR ( Por otro lado, en modelos animales, se ha propuesto que la actividad Xantina Oxido-Reductasa (XOR) dependiente de nitrito, puede ser responsable de la producción de NO bajo determinadas condiciones (Zhang et al, 1997;Millar et al, 1998;Zhang et al, 1998). En Arabidopsis, la XOR esta codificada por dos genes, XOR1 (At4g34890) y XOR2 (At4g34900) con una alta homología (Hesberg et al, 2004). Como ha sido postulado que este tipo de actividad puede ser también responsable de la producción de NO en plantas (del Rio et al, 2002;del Rio et al, 2004), obtuvimos mutantes de las colecciones públicas de inserciones de T-DNA nulos en ambos genes.…”

“…Esta es, de nuevo, una enzima bi-funcional que se puede encontrar en la forma Xantina Deshidrogenasa (XDH) o en la forma, dependiente de O 2 , Xantina Oxidasa (XO). Mientras que la forma XDH tiene preferencia por NAD + sobre O 2 como aceptor de electrones, la forma XO tiene una actividad estrictamente dependiente de O 2 (Hesberg et al, 2004). Estas dos formas de la enzima se pueden interconvertir, reversiblemente mediante oxidaciones de residuos de cisteína (Stirpe y Della Corte 1969), o irreversiblemente por proteolisis (Amaya et al, 1990).…”

“…Así, si bien la actividad XOR o la propia proteína, participa de alguna manera en la producción de NO, sus mutantes deberían tener una menor acumulación de NO posiblemente en hojas o tejidos fotosintéticos más acentuada que en raíces. Sin embargo, la actividad XOR se regula también a nivel post-traduccional mediante la activación del holoenzima de manera que los niveles de proteína no siempre se correlacionan bien con los niveles de actividad (Hesberg et al, 2004). De hecho, pese a una diferencia importante en la transcripción de XOR1 y XOR2 en raíces y en hojas, la diferencia en actividad XOR no es tan importante.…”