The biochemistry of flavone C-glycosides in cereals

类黄酮是在所有植物中发现的具有重要生物学意义的天然产物，越来越多地被认为是健康饮食中的关键营养成分。作为主要的粮食作物，主要谷物(小麦、水稻、玉米)对我们的饮食有很大的贡献，它们都含有一组不寻常的生物活性类黄酮，它们通过碳-碳(C-C)键与糖分子相连。这与大多数类黄酮相反，后者通过更不稳定的碳氧(醚)键连接到糖上。这两类糖苷之间的一个重要区别是，当乙醚糖苷被摄入时很容易被水解，而C-糖苷则不是。通过将黄酮类化合物的生物活性与糖成分的生物活性结合起来，自然界衍生出了一些异常具有生物活性的次生代谢物，这些次生代谢物在宿主植物和摄取它们的动物中扮演着不同的角色。在植物中，黄酮糖苷具有抗氧化剂和紫外线吸收色素的活性，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，它们已经被证明可以对抗炎症和氧化损伤，尽管就像许多植物次产品一样，在高剂量下，它们可能具有有害活动。基于它们在主要粮食作物中的相对丰度和高生物活性，令人惊讶的是，与黄酮类O-糖苷的合成不同，人们对植物中产生C-糖苷的途径研究很少。为了研究植物中调节小分子生物活性的生物转化反应，本课题组最近从水稻和小麦中分离纯化了黄酮类C-糖基转移酶(CGTS)，并鉴定了编码相应酶的基因。我们现在建议研究CGTS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于产生黄酮糖苷。CGT与为其提供底物的酶(黄烷酮2-羟基酶)和第二种蛋白质的组织尤其重要，第二种蛋白质将C-糖基化的中间体转化为生物活性的黄酮-C-糖苷，因为这一小型途径代表了在谷物和其他主要作物中生产类黄酮的一种基本上不为人所知的方法。我们将结合有机化学、生物化学和代谢工程来研究CGT及其相关酶在水稻和小麦中的功能。小麦对英国来说是最具战略价值的作物，而水稻更好的特性和更简单的遗传学使后者成为应用基因组工具研究其生物化学的非常有用的模型，然后将成果应用于其他谷物。首先，我们将通过研究CGT的酶机制来确定CGT是如何催化C-C键的形成的。然后，我们将寻找负责将第二个C-共轭糖添加到类黄酮中的其他CGT活性。然后，CGT活性将被设置为它在植物中如何受到调节的背景。我们最近确定，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中黄酮类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调节的。然后，这些研究将为研究CGT如何与酶一起工作奠定基础，这些酶为CGT提供底物，并通过在微生物和植物宿主细胞中共表达该途径的组成部分并监测它们在体内产生的代谢物来处理其产品。该计划的目标是将我们对黄酮C-糖基途径的生物化学的了解提高到这样的水平，即我们可以合理地控制它们在植物中的积累，或者在可发酵的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.