ENZYMES OF THIAMIN METABOLISM

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 焦磷酸硫胺素是细胞内发现的维生素B1的活性辅因子形式，是人类必需的维生素。虽然人类不能生物合成这种化合物，但大多数植物和细菌都能产生硫胺素。硫胺素生物合成的途径在细菌与植物和真菌之间变化，细菌利用具有轻微变化的途径，植物和真菌依赖于不同的途径。也有可能回收硫胺素和硫胺素的降解形式。在真菌和植物系统中，THI 1和THI 4分别负责形成噻唑前体二磷酸腺苷-5-（-乙基）-4-甲基噻唑-2-羧酸。THI 6是一种双功能酶，催化噻唑部分的磷酸化，并将该反应的产物与嘧啶部分偶联以产生硫胺素单磷酸。在酵母中，THI 20是第二种双功能酶，既水解硫胺素又磷酸化嘧啶部分。 在细菌中，噻唑和嘧啶部分分别形成，然后依靠许多酶连接在一起。ThiE，硫胺素磷酸合酶，负责这种偶联反应。4-氨基-5-羟甲基-2-甲基嘧啶磷酸（HMP-P）合酶（ThiC）是一种铁硫簇蛋白，负责5-氨基咪唑核糖核苷酸（AIR）的剧烈重排以形成HMP-P。 硫胺素生物合成中的一个重要步骤是将硫原子掺入不断生长的噻唑部分。在细菌中的噻唑生物合成中，这是通过硫载体蛋白ThiS的活化和与第二种蛋白ThiF合作在ThiS的羧基末端形成硫酯来实现的。一旦硫酯在ThiS上形成，硫原子就通过ThiG和ThiO/ThiH掺入噻唑部分。 最近，一个新的硫酸盐同化途径Wolinella succinogenes已被确定，包含四个酶，具有一级结构的硫转移酶的硫胺素生物合成的相似性。这些酶在W.琥珀酸酯酶包括ThiS样蛋白、ThiF样蛋白、QBSD样蛋白和推定的O-乙酰高丝氨酸巯基化酶（OAHS），其可能参与硫化物向活化的WsThiS样蛋白的转移。 除了硫胺素代谢酶，我们正在研究一种重要的有毒硫胺素抗代谢物，称为bacimethrin。 它是由一些革兰氏阳性细菌产生的分子，其竞争硫胺素生物合成途径的内源性底物以产生硫胺素衍生物。 已经在肉毒梭菌中鉴定了负责杆菌菊酯生物合成的基因簇，并且已经证实了这些基因产物中的每一个的功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Thiamin pyrophosphate, the active cofactor form of vitamin B1 found within the cell, is an essential vitamin for humans. Although humans cannot biosynthesize this compound, most plants and bacteria are capable of producing thiamin. The pathways for thiamin biosynthesis vary between bacteria, which utilize a pathway with slight variations, and plants and fungi, which rely upon a different pathway. It is also possible for thiamin and degraded forms of thiamin to be salvaged. In fungal and plant systems, THI1 and THI4 respectively, are responsible for the formation of the thiazole precursor adenosine diphospho-5-(-ethyl)-4-methylthiazole-2-carboxylic acid. THI6, a bifunctional enzyme, catalyzes the phosphorylation of the thiazole moiety and couples the product of this reaction to the pyrimidine moiety to yield thiamin monophosphate. In yeast, THI20 is a second bifunctional enzyme that both hydrolyzes thiamin and phosphorylates the pyrimidine moiety. In bacteria, the thiazole and pyrimidine moieties are formed separately and then joined together relying on many enzymes. ThiE, thiamin phosphate synthase, is responsible for this coupling reaction. 4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase, or ThiC, is an iron-sulfur cluster protein responsible for the dramatic rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P. An important step in the biosynthesis of thiamin is the incorporation of an atom of sulfur into the growing thiazole moiety. In thiazole biosynthesis in bacteria, this is achieved by the activation of a sulfur carrier protein, ThiS, and the formation of a thioester at the carboxy terminus of ThiS in cooperation with a second protein, ThiF. Once the thioester has been formed on ThiS, the sulfur atom is then incorporated into the thiazole moiety via ThiG and ThiO/ThiH. Recently, a new sulfate assimilation pathway from Wolinella succinogenes has been identified, containing four enzymes which have primary structure similarity to the sulfur transfer enzymes of thiamin biosynthesis. These enzymes in W. succinogenes include a ThiS-like protein, a ThiF-like protein, a QBSD-like protein, and a putative O-acetylhomoserine sulfydrylase (OAHS), which might be involved in the transfer of sulfide to the activated WsThiS-like protein. In addition to enzymes of thiamin metabolism, we are studying an important toxic thiamin antimetabolite called bacimethrin. It is a molecule produced by some gram-positive bacteria that outcompetes the endogenous substrates of the thiamin biosynthetic pathway to produce a thiamin derivative. The gene cluster responsible for bacimethrin biosynthesis has been identified in Clostridium botulinum and the functions for each of these gene products has been confirmed.