Membrane Protein Research Group, Department of Biochemistry, University of Alberta, 474 Medical Sciences Building, Edmonton, Alberta, T6G 2H7, Canada.
Plant Mol Biol. 2010 Feb;72(3):301-10. doi: 10.1007/s11103-009-9570-2. Epub 2009 Nov 14.
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.
拟南芥黄嘌呤脱氢酶 AtXDH1 是嘌呤降解中的关键酶，它将次黄嘌呤氧化为黄嘌呤，将黄嘌呤氧化为尿酸。这些底物释放的电子要么转移到 NAD(+)，要么转移到分子氧，从而分别产生 NADH 或超氧阴离子。通过另一种活性，AtXDH1 能够氧化 NADH，同时形成 NAD(+)和超氧阴离子。在这里，我们证明与作为底物的黄嘌呤的比活性相比，重组 AtXDH1 用 NADH 作为底物的比活性大约高 15 倍，同时超氧阴离子的产生增加了一倍。观察到 NAD(+)抑制 AtXDH1 的 NADH 氧化酶活性，而 NADH 抑制 NAD(+)-依赖的黄嘌呤氧化表明 NAD(+)和 NADH 竞争相同的结合位点，并且两种亚活性不会同时表达。相反，每种亚活性都由特定条件决定，例如底物和辅助底物的可用性，这允许通过 AtXDH1 调节超氧阴离子的产生。由于 AtXDH1 在迄今为止研究的所有黄嘌呤脱氢酶蛋白中表现出最显著的 NADH 氧化酶活性，我们的结果表明，AtXDH1 特别通过其 NADH 氧化酶活性也是体内超氧阴离子的有效产生者。
