ENZYMES OF THIAMIN METABOLISM

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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，是人类必不可少的维生素。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不同，细菌利用的途径略有不同，植物和真菌依赖于不同的途径。硫胺素和降解形式的硫胺素也有可能被挽救。在真菌和植物系统中，THI1和THI4分别负责噻唑前体腺苷diphospho-5-(-ethyl)-4-methylthiazole-2-carboxylic酸的形成。THI6是一种双功能酶，它催化噻唑部分的磷酸化，并将反应产物与嘧啶部分偶联，生成单磷酸硫胺素。在酵母中，THI20是第二种双功能酶，既能水解硫胺素，又能使嘧啶部分磷酸化。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。硫胺素磷酸合成酶负责这一偶联反应。4-氨基-5-羟甲基-2-甲基嘧啶磷酸(HMP-P)合成酶，简称THIC，是一种铁硫簇蛋白，负责5-氨基咪唑核苷酸(AIR)的戏剧性重排形成HMP-P。 硫胺素生物合成的一个重要步骤是将一个硫原子加入到生长中的噻唑部分中。在细菌中的噻唑生物合成中，这是通过激活硫载体蛋白This，并在其羧基末端与第二种蛋白质ThiF合作形成硫酯来实现的。一旦硫酯在其上形成，硫原子就通过硫代和硫代/硫代结合到噻唑部分中。最近，从产琥珀酸狼菌中发现了一种新的硫酸盐同化途径，它含有四种与硫胺素生物合成的硫转移酶具有一级结构相似的酶。这些酶包括一个This-like蛋白、一个ThiF-like蛋白、一个QBSD-like蛋白和一个可能参与硫化物向激活的WsThiS-like蛋白转移的O-乙酰高丝氨酸硫化酶(OAHS)。 除了硫胺素代谢的酶，我们正在研究一种重要的毒性硫胺素抗代谢物质，称为杆菊酯。它是一种由一些革兰氏阳性细菌产生的分子，它比硫胺素生物合成途径的内源底物竞争产生硫胺素衍生物。在肉毒梭菌中，已经鉴定了负责杆菊酯生物合成的基因簇，并确认了每个基因产物的功能。

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.