Structure and reaction mechanism of benzylsuccinate synthase and its activating enzyme

苄基琥珀酸合酶及其激活酶的结构和反应机制

• 批准号: 71841172
• 负责人: Professor Dr. Johann Heider
• 金额: --
• 依托单位: Laboratory for Microbial Biochemistry
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/71841172?language=en
• 关键词: Structure; reaction; mechanism; benzylsuccinate; synthase

All known organisms capable of anaerobic toluene metabolism initiate this pathway by addition of the methyl group of toluene to a fumarate cosubstrate to yield (R)-benzylsuccinate. This reaction represents a new biochemical principle and is catalysed by a glycyl-radical-enzyme, benzylsuccinate synthase (BSS). This enzyme has since become a model for enzymes catalysing analogous fumarate additions in anaerobic metabolic pathways of other hydrocarbons, including alkanes. Benzylsuccinate synthase contains a free radical on a conserved glycine residue, which is inserted by a separate, S-adenosylmethionine- dependent activating enzyme. The catalytic mechanism is proposed to involve the formation of an enzyme-bound substrate-radical from toluene, which then reacts with fumarate to a product-radical. Unlike other known glycyl-radical-enzymes, BSS contains additional subunits and [Fe4S4]-clusters whose functions are not yet understood. This proposal is intended to investigate the structure-function relationship of the benzylsuccinate synthase reaction mechanism and follows the following major aims: [1] solving the crystal structure of non-activated recombinant benzylsuccinate synthase. We have already attained a resolvation of the structure at 3.4 Ǻ, which promises to yield the elucidation of the structure during the next funding period. [2] further investigations on the reaction mechanism of BSS, providing a model for similar enzymes involved in anaerobic catabolism of other hydrocarbons, and [3] crystallising and characterising the activating enzyme and the activation reaction.

所有已知的能够厌氧甲苯代谢的生物都通过将甲苯的甲基加到富马酸共底物上产生(R)-苄基琥珀酸盐来启动这一途径。该反应代表了一种新的生化原理，由甘酰基自由基酶苄基琥珀酸合成酶（BSS）催化。这种酶已经成为催化其他碳氢化合物（包括烷烃）厌氧代谢途径中类似富马酸添加的酶的模型。苄基琥珀酸合酶在一个保守的甘氨酸残基上含有一个自由基，该残基由一个独立的s -腺苷甲硫氨酸依赖的激活酶插入。提出了催化机制涉及到从甲苯形成酶结合的底物自由基，然后与富马酸盐反应生成产物自由基。与其他已知的甘酰基自由基酶不同，BSS含有额外的亚基和[Fe4S4]簇，其功能尚不清楚。本课题旨在研究苄基琥珀酸合酶反应机理的结构-功能关系，主要目的如下：[1]解决非活化重组苄基琥珀酸合酶的晶体结构。我们已经在3.4 Ǻ中获得了结构的解析，它有望在下一个资助期内对结构进行阐明。[3]进一步研究了BSS的反应机理，为参与其他碳氢化合物厌氧分解代谢的类似酶提供了模型，[3]结晶并表征了活化酶和活化反应。