Hesberg Christine, Hänsch Robert, Mendel Ralf R, Bittner Florian
Department of Plant Biology, Technical University of Braunschweig, 38023 Braunschweig, Germany.
J Biol Chem. 2004 Apr 2;279(14):13547-54. doi: 10.1074/jbc.M312929200. Epub 2004 Jan 15.
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.
对来自拟南芥的黄嘌呤脱氢酶进行了分子和生化水平的分析。大多数其他生物体似乎仅拥有一个黄嘌呤脱氢酶基因，而拟南芥拥有两个串联排列的基因，间隔704个碱基对。cDNA以及AtXDH1和AtXDH2蛋白的总体一致性为93%，并与其他生物体的黄嘌呤脱氢酶具有高度同源性。AtXDH2 mRNA组成型表达，而在干旱、盐胁迫、寒冷和自然衰老等各种胁迫下，以及脱落酸处理后，观察到AtXDH1转录水平的变化。转录本的改变不一定导致黄嘌呤脱氢酶活性的变化。盐处理对黄嘌呤脱氢酶活性没有影响，冷胁迫导致活性降低，但干燥和衰老导致叶片中活性大幅增加。由于AtXDH1可能是拟南芥中更重要的同工酶，因此将其在毕赤酵母中表达、纯化并用于生化研究。AtXDH1蛋白是一个约300 kDa的同型二聚体，由150 kDa的相同亚基组成。与其他生物体的黄嘌呤脱氢酶一样，AtXDH1以次黄嘌呤和黄嘌呤作为主要底物，并受到别嘌呤醇的强烈抑制。AtXDH1可以被纯化的钼辅因子硫化酶ABA3激活，ABA3将无活性的脱硫酶转化为有活性的含硫酶。最后发现，AtXDH1是一种严格的脱氢酶而非氧化酶，但能够产生超氧自由基，这表明除了嘌呤分解代谢外，它可能还参与对需要活性氧的各种胁迫的响应。
