The biochemistry of flavone C-glycosides in cereals

黄酮类化合物是在所有植物中发现的重要的生物天然产物，在健康饮食中越来越被认为是关键的营养保健品。作为主食作物，主要谷物（小麦、大米、玉米）对我们的饮食做出了重要贡献，它们都含有一组不同寻常的生物活性类黄酮，这些类黄酮通过碳-碳（C-C）键与糖分子相连。这与大多数类黄酮形成对比，类黄酮通过更不稳定的碳氧（醚）键与糖相连。这两类糖苷之间的一个重要区别是，乙醚糖苷在摄入时很容易水解，而c -糖苷则不然。通过将黄酮类化合物的生物活性与糖成分的生物活性相结合，自然界衍生出了一些具有不同寻常的生物活性的次生代谢物，这些代谢物在宿主植物和食用它们的动物中都发挥着各种作用。在植物中，黄酮- c -糖苷具有抗氧化剂和吸收紫外线色素的活性，并调节与微生物、昆虫和其他植物的相互作用。作为我们饮食中的微量成分，它们已被证明可以抵消炎症和氧化损伤，尽管与许多植物次生产物一样，高剂量时它们可能具有有害活性。基于它们在主要粮食作物中的相对丰度和高生物活性，因此令人惊讶的是，与类黄酮- o -糖苷的合成非常清楚不同，植物中负责生产c -糖苷的途径很少被研究。由于对植物中调节小分子生物活性的生物转化反应感兴趣，我们的团队最近从水稻和小麦中纯化了类黄酮c -糖基转移酶（cgt），并鉴定了编码这些酶的基因。我们现在建议研究cgt如何催化这种不寻常的活性，以及它们如何与其他类黄酮代谢酶一起作用以产生黄酮c -糖苷。CGT与为其提供底物的酶（黄酮2-羟化酶）以及将c -糖基化中间体转化为生物活性黄酮- c -糖苷的第二种蛋白质的组织尤其重要，因为这种迷你途径代表了谷物和其他主要作物中大部分未被认识的生产类黄酮的方式。我们将运用有机化学、生物化学和代谢工程相结合的方法来研究CGT及其相关酶在水稻和小麦中的功能。鉴于小麦是英国最具战略价值的作物，水稻的更好的特征和更简单的遗传使后者成为应用基因组工具研究其生物化学的一个非常有用的模型，然后将结果应用于其他谷物。首先，我们将通过研究CGT酶的机制来确定CGT如何催化C-C键的形成。然后，我们将寻找其他CGT活性，这些活性负责向类黄酮添加第二个c -共轭糖。然后将CGT活性置于植物如何调节的背景下。我们最近确定了一组被称为除草剂安全剂的农用化学品选择性地控制小麦黄酮c -糖苷的积累。我们现在可以使用这些化学工具来研究类黄酮代谢的这一分支是如何被调节的。这些研究将通过在微生物和植物宿主细胞中共同表达该途径的组成部分，并监测它们在体内产生的代谢物，为检验CGT如何与为其提供底物和加工其产物的酶一起工作奠定基础。该项目的目标是将我们对黄酮c -糖基途径的生物化学理解提高到一个水平，使我们能够合理地控制它们在植物中的积累，或者在可发酵微生物中产生这些生物活性代谢物。

Flavonoids are biologically important natural products found in all plants which are increasingly recognized as key nutraceuticals in a healthy diet. As staple food crops, the major cereals (wheat, rice, maize) make major contributions to our diet and all contain an unusual group of bioactive flavonoids which are joined to a sugar molecule through a carbon-carbon (C-C) bond. This is in contrast to the majority of flavonoids, which are attached to sugars through more labile carbon-oxygen (ether) linkages. An important distinction between the two classes of glycosides, is that whereas the ether glycosides are readily hydrolysed when they are ingested, the C-glycosides are not. By combining the biological activity of the flavonoids with that of the sugar component, nature has derived some unusually bioactive secondary metabolites which play a variety of roles in both the host plant and in animals which ingest them. In plants, flavone-C-glycosides have activities as antioxidants and UV-absorbing pigments and regulate interactions with microbes, insects and other plants. As trace components in our diet, they have been demonstrated to counteract inflammation and oxidative damage, though as is the case with many plant secondary products, at high doses they can have deleterious activities. Based on their relative abundance in staple food crops and high bioactivity it is therefore surprising that unlike the flavonoid-O-glycosides whose synthesis is very well understood, that the pathways responsible for producing C-glycosides in plants have been little studied. With an interest in biotransformation reactions in plants which regulate the bioactivity of small molecules, our group has recently purified flavonoid C-glycosyltransferases (CGTs) from both rice and wheat and identified the genes encoding the respective enzymes. We now propose to study how CGTs catalyse this unusual activity and how they function with other enzymes of flavonoid metabolism to produce flavone C-glycosides. The organization of the CGT with the enzyme (flavanone 2-hydroxylase), which provides it with its substrate, and a second protein which converts the C-glycosylated intermediate to the bioactive flavone-C-glycoside is particularly important, as this mini-pathway represents a largely unrecognized means of producing flavonoids in cereals and other major crops. We will use a combination of organic chemistry, biochemistry and metabolic engineering to study the functioning of the CGT and its associated enzymes in rice and wheat. Whereas wheat is the crop of most strategic value to the UK, the better characterized and simpler genetics of rice make the latter a very useful model to apply genomic tools to study its biochemistry, with the outputs then being applied to other cereals. Firstly we will determine how the CGT catalyses the formation of C-C bonds by investigating the mechanism of the enzyme. We will then look for other CGT activities which are responsible for adding a second C-conjugated sugar to the flavonoid. The CGT activity will then be set into the context of how it is regulated in plants. We have recently determined that a group of agrochemicals called herbicide safeners selectively control flavone C-glycoside accumulation in wheat. We can now use these chemical tools to study how this branch of flavonoid metabolism is regulated. These studies will then set the scene for examining how the CGT works with the enzymes which supply it with substrate and process its product by co-expressing the component parts of the pathway in microbial and plant host cells and monitoring the metabolites they produce in vivo. The objective of the programme is to take our understanding of the biochemistry of the flavone C-glycosyl pathway to a level where we can either rationally manipulate their accumulation in plants, or produce these bioactive metabolites in fermentable microbes.