Structure and reaction mechanism of benzylsuccinate synthase and its activating enzyme

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来产生(R)-苄基琥珀酸酯来启动这一途径。该反应代表了一种新的生化原理，由甘氨酰自由基酶--丁二酸苄酯合成酶(BSS)催化。自那以后，这种酶已经成为在包括烷烃在内的其他碳氢化合物的厌氧代谢途径中催化类似富马酸加成的酶的模型。丁二酸苄酯合成酶在一个保守的甘氨酸残基上含有一个自由基，该甘氨酸残基由一个单独的S-腺苷蛋氨酸依赖的活化酶插入。其催化机理是由甲苯生成酶结合的底物自由基，再与富马酸反应生成产物自由基。与其他已知的甘氨酰自由基酶不同，BSS含有额外的亚基和[Fe4S4]-簇，其功能尚不清楚。本研究旨在探讨琥珀酸苄酯合成酶反应机理的构效关系，主要目的如下：[1]解决未活化的重组琥珀酸苄酯合成酶的晶体结构问题。我们已经在3.4GMT获得了结构的解析，这有望在下一个资助期内得到结构的阐明。[2]进一步研究BSS的反应机理，为类似酶参与其他碳氢化合物的厌氧分解代谢提供模型，以及[3]活化酶和活化反应的结晶和表征。

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.