Characterisation of the xanthine oxidase protein family in Arabidopsis: biochemistry and physiological importance for the synthesis of phytohormones, ureides, and reactive oxygen species

黄嘌呤氧化酶家族由醛氧化酶和黄嘌呤脱氢酶两种蛋白质组成，不同的植物物种编码不同数量的同工酶。在拟南芥中，存在AAO1-AAO4四种AO亚型，它们既形成同源二聚体，也形成异源二聚体。虽然AA03被认为是脱落酸合成的关键酶，但其他亚型的生理功能大多还不清楚，也知之甚少。然而，根据底物的特性，假设AAO1和/或AAO2催化吲哚乙醛氧化成吲哚乙酸，从而它们可以参与几种可能的吲哚乙酸合成方法之一。虽然AAO4被认为在硫代葡萄糖苷的合成中发挥作用，但对此仍缺乏令人信服的证据。因此，详细研究和鉴定醛氧化酶亚型AAO1、2和4的生理功能是本项目的一个方面。在拟南芥中，存在两个XDH基因，其中AtXDH2代表假基因，AtXDH1是嘌呤分解的关键酶，催化次黄嘌呤通过黄嘌呤氧化成尿酸。XDH是AO的进化祖先，因此，这两种酶都有某些共同的特性：两种酶都能够将底物衍生的电子转移到分子氧中，同时在XDH的情况下形成超氧化物，在AO的情况下同时形成超氧化物和过氧化氢。此外，我们能够证明这两种酶都具有内在的NADH氧化酶活性，这同样与超氧化物的形成有关。有趣的是，所有与AA03和/或AtXDH1活性增加相关的生理条件都同样需要增加活性氧物种的水平(如干旱胁迫、衰老)。由于AO和/或XDH产生的活性氧物种的生理意义甚至还没有被研究过，这将是这个提议的另一个主题。在我们的初步工作中，AA03和AtXDH1已经被证明受到泛素化的调节，泛素化能力的丧失导致这些酶的积累和过早衰老。除了相应的泛素化动机外，AA03和AtXDH1也具有明显的苏莫化动机，后者在早期的研究中没有被考虑。由于在其他的AO亚型中也发现了这样的动机，我们想要阐明是否所有或一些黄嘌呤氧化酶家族的酶都在这些翻译后修饰之下，如果是的话，是出于什么生理原因。最后，通过使用重组蛋白，我们将分析AO和XDH蛋白的几个生化特性，特别是底物的特性和一氧化氮的可能产生，这是有争议的讨论了很长时间。

The xanthine oxidase family comprises aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) proteins, with different plant species encoding a varying number of isoenzymes. In Arabidopsis four AO isoforms, AAO1-AAO4, are existing, which form homodimers as well as heterodimers. While AAO3 is well accepted as a key enzyme in abscisic acid synthesis, the physiological functions of the other isoforms are mostly uncharacterised and only poorly understood. Yet, based on substrate specificities it is assumed that AAO1 and/or AAO2 catalyse the oxidation of indole acetaldehyde to indole acetic acid, by which they could be involved in one out of several possible ways of indole acetic acid synthesis. Although AAO4 is assumed to play a role in glucosinolate synthesis, convincing evidence for this is still lacking. Thus, it is one aspect of the present project to study in detail and to identify the physiological functions of the aldehyde oxidase isoforms AAO1, 2, and 4.In Arabidopsis, two XDH genes are exisiting with AtXDH2 representing a pseudogene and AtXDH1 being a key enzyme in purine breakdown that catalyses the oxidation of hypoxanthine via xanthine to uric acid. XDH is the evolutionary ancestor of AO and thus, both enzymes share certain properties: Both enzymes are able to transfer substrate-derived electrons to molecular oxygen with simultaneous formation of superoxides in case of XDH and superoxides and hydrogen peroxide in case of AO. Moreover, we were able to show that both enzymes harbor an intrinsic NADH oxidase activity which is likewise associated with the formation of superoxides. Interestingly, all physiological conditions associated with increased activities of AAO3 and/or AtXDH1 are likewise characterised by a requirement for increased levels of reactive oxygen species (e.g. drought stress, senescence). Since a physiological significance of reactive oxygen species produced by AO and/or XDH has not even been investigated as yet, this will be another subject of this proposal.In our preliminary work, AAO3 and AtXDH1 have been demonstrated to be regulated by ubiquitination, with a loss of ubiquitination capacity resulting in accumulation of these enzymes and premature senescence. Besides corresponding ubiquitination motives, AAO3 and AtXDH1 also possess pronounced sumoylation motives, with the latter not having been considered in earlier studies. Since such motives are also found in other AO isoforms, we wish to elucidate whether all or some enzymes of the xanthine oxidase family underly these post-translational modifications, and if so, for what physiological reason.Finally, by using recombinant proteins we will analyse several biochemical properties of AO and XDH proteins with a special focus on the characterisation of substrate specificities and a possible production of nitric oxide, which is controversially discussed for a long time.