权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

拟南芥中黄嘌呤氧化酶蛋白家族的表征：对植物激素、酰脲和活性氧合成的生物化学和生理重要性

基本信息

批准号：
234281575
负责人：
Dr. Christian Gehl, since 10/2015
金额：
--
依托单位：
Julius Kühn-Institut (JKI) - Bundesforschungsinstitut für Kulturpflanzen
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2014-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/234281575?language=en
关键词：
Characterisation xanthine oxidase protein family

项目摘要

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.

黄嘌呤氧化酶家族包括醛氧化酶（AO）和黄嘌呤脱氢酶（XDH）蛋白，不同的植物物种编码不同数量的同工酶。在拟南芥中，存在四种AO同工型AAO 1-AAO 4，它们形成同源二聚体以及异源二聚体。虽然AAO 3被公认为脱落酸合成中的关键酶，但其他同工型的生理功能大多未被表征，并且仅知之甚少。然而，基于底物特异性，假设AAO 1和/或AAO 2催化吲哚乙醛氧化为吲哚乙酸，通过该氧化，它们可能参与吲哚乙酸合成的几种可能方式之一。虽然AAO 4被认为在芥子油苷合成中起作用，但仍然缺乏令人信服的证据。因此，深入研究和鉴定醛氧化酶AAO 1、2和4的生理功能是本研究的一个方面。在拟南芥中，存在两个XDH基因，其中AtXDH 2为假基因，AtXDH 1为嘌呤分解的关键酶，催化次黄嘌呤经黄嘌呤氧化为尿酸。XDH是AO的进化祖先，因此，两种酶都具有某些特性：两种酶都能够将底物衍生的电子转移到分子氧，同时在XDH的情况下形成超氧化物，在AO的情况下形成超氧化物和过氧化氢。此外，我们能够表明，这两种酶都具有固有的NADH氧化酶活性，这同样与超氧化物的形成有关。有趣的是，与AAO 3和/或AtXDH 1活性增加相关的所有生理条件同样以需要增加的活性氧水平为特征（例如干旱胁迫、衰老）。由于AO和/或XDH产生的活性氧的生理意义尚未研究，这将是本提案的另一个主题。在我们的初步工作中，AAO 3和AtXDH 1已被证明是由泛素化调节，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，AAO 3和AtXDH 1也具有明显的SUMO化动机，后者在早期的研究中没有被考虑。由于这样的动机也发现在其他AO亚型，我们希望阐明是否所有或一些酶的黄嘌呤氧化酶家族的基础上这些翻译后修饰，如果是这样的话，是什么生理原因。最后，通过使用重组蛋白，我们将分析几个生化特性的AO和XDH蛋白与一个特别关注的特点，底物特异性和可能的生产一氧化氮，这是一个争议性很长时间的问题。