Thiamin Metabolism in Plants: Elucidation of the 4-Methyl-5-(b-hydroxyethyl)Thiazole Phosphate (HET) Biosynthetic Pathway in Arabidopsis

焦磷酸硫胺（TPP）是由两种主要中间体4-甲基-5-（b-羟乙基）噻唑磷酸（HET）和2-甲基-4-氨基-5-羟甲基嘧啶焦磷酸（HMP）缩合而成，是所有生物生存所必需的酶辅因子。虽然TPP仅由细菌、真菌和光合生物合成，但动物和人类必须从饮食中获得硫胺素。虽然植物是膳食中硫胺素的主要来源，但令人惊讶的是，人们对植物中硫胺素合成的大多数方面知之甚少。TPP的HET中间体尤其如此。已知的heet生物合成大部分来自微生物的遗传和生化研究，其中这种化合物是使用不同的酶机制从不同的合成前体中提取的。虽然先前的研究表明，HET是在大肠杆菌中合成的，其中脱氧-5-磷酸木醛糖（DXP）是前体，但Shintani实验室的研究表明，植物可以在没有DXP的情况下合成HET。这一结果表明，植物可能拥有一种替代HET的途径，该途径可以替代或补充先前描述的“大肠杆菌样”途径。本研究的总体目标是使用分子和生化方法的结合来确定导致植物中HET形成的生物合成途径。为了实现这一目标，将进行以下研究目标：1)确定导致植物中硫胺素生物合成的生物合成前体；2)确定酵母Thi4蛋白的植物同源物在HET生物合成中的作用；3)鉴定和功能分析其他拟南芥HET生物合成基因。人体缺乏硫胺素（维生素B1）可导致脚气病，表现为严重的神经紊乱和普遍的消瘦现象。这种疾病主要与发展中国家的贫困人口有关，他们的饮食主要以精米或漂白小麦粉等精加工谷物产品为生。解决人类维生素缺乏症的可持续办法是增加世界上内源性人口通常食用的主要粮食作物的营养成分。通过对这类作物进行基因工程以增加硫胺素，应该有可能对全球人口的营养需求产生积极影响。不幸的是，这种努力的主要障碍是目前缺乏有关植物中硫胺素生物合成的知识。通过这些研究获得的生物合成知识的增加将对合理设计用于提高硫胺素水平以改善人类和动物营养的作物具有重要意义。

Thiamin pyrophosphate (TPP), which is formed through the condensation of two major intermediates, 4-methyl-5-(b-hydroxyethyl)thiazole phosphate (HET) and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP), is an essential enzyme co-factor required for the viability of all organisms. While TPP is only synthesized by bacteria, fungi and photosynthetic organisms, animals and humans must derive TPP from dietary sources of thiamin. Although plants represent a major source of dietary thiamin, surprisingly little is understood about most aspects of its synthesis in plants. This is particularly true for the HET intermediate of TPP. Most of what is known about HET biosynthesis has been derived from genetic and biochemical studies in microorganisms where this compound is derived from different synthetic precursors using different enzymatic machinery. Although a previous study suggested that HET is synthesized as described for E. coli, where deoxy-xylulose-5-phosphate (DXP) is a precursor, the Shintani lab has performed studies showing that plants can synthesize HET in the absence of DXP. This result suggests that plants may possess an alternative pathway to HET that exists in place of or in addition to the previously described "E. coli-like" pathway. The overall goal of this research is to use a combination of molecular and biochemical approaches to determine the biosynthetic pathway(s) leading to formation of HET in plants. To achieve this goal the following research objectives will be performed: 1) Determine the biosynthetic precursors leading to thiamin biosynthesis in plants, 2) Determine the role of the plant orthologue of the yeast Thi4 protein in HET biosynthesis, 3) Identify and functionally analyze additional Arabidopsis HET biosynthetic genes. Thiamin (Vitamin B1) deficiencies in humans can lead to a condition known as Beriberi that is manifested by severe neurological disorders and a general wasting phenomenon. This disease is primarily associated with poverty-stricken populations of developing countries whose diets subsist primarily of polished grain products such as polished rice or bleached wheat flour. A sustainable solution to thiamin deficiencies in humans would be to increase the nutritional content of staple food crops that endogenous populations of the world commonly consume. By genetic engineering such crops for increased thiamin, it should be possible to positively impact the nutritional needs of the global population. Unfortunately, the major impediment to such an effort is a current lack of knowledge pertaining to the biosynthesis of thiamin in plants. The increased biosynthetic knowledge obtained through these studies will be important for the rational design of crops engineered for elevated thiamin levels for improved human and animal nutrition.