A Mechanistic Model of the Cysteine Synthase Complex
A Mechanistic Model of the Cysteine Synthase Complex
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DOI：
10.1016/j.jmb.2008.08.075
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发表时间：
2009-02-13
影响因子：
5.6
通讯作者：
Wade, Rebecca C.
中科院分区：
文献类型：
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作者：
Feldman-Salit, Anna;Wirtz, Markus;Wade, Rebecca C.
植物和细菌吸收无机硫并将其结合到有机化合物中，如氨基酸半胱氨酸。半胱氨酸生物合成涉及双酶复合物，半胱氨酸合酶（CS）复合物。CS复合物由丝氨酸乙酰转移酶（SAT）和O-乙酰丝氨酸-（巯基）-裂解酶（OAS-TL）组成。虽然它是实验上已知的CS复合物的形成影响半胱氨酸的生产，CS复合物的确切的生物功能，组成酶的相互调节的机制和复合物的结构仍然知之甚少。在这里，我们使用对接技术来构建一个模型的CS复杂的线粒体拟南芥。通过比较技术对酶的三维结构进行建模。SAT的C-末端，在模板结构中缺失，但对CS形成至关重要，从头建模。通过刚体布朗动力学模拟，生成了SAT和OAS-TL的扩散相遇复合体。通过在布朗动力学模拟过程中加入实验约束，我们确定了与实验一致的复合物。通过分子动力学模拟对选定的遭遇复合物进行了改进，以生成结合复合物的结构。我们发现，虽然化学计量比的6个OAS-TL二聚体的一个SAT六聚体在CS复杂的几何是可能的，结合能计算表明，与实验一致，只有两个OAS-TL二聚体的比例，一个SAT六聚体是更有可能的。在OAS-TL的计算突变的残基是实验上显着的CS形成阻碍了协会的酶由于一个不太有利的静电结合自由能。由于A. thaliana的SAT和OAS-TL在大肠杆菌中表达，并进行跨种结合试验。coli和A. Thaliana已经开发了。结果表明，半胱氨酸产量的减少可能是由于A. thaliana OAS-TL与E. coli SAT.所提出的酶及其复合物的模型提供了CS络合的机理见解。(C)2008爱思唯尔有限公司保留所有权利。
Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is composed of the enzymes serine acetyl transferase (SAT) and O-acetylserine-(thiol)-lyase (OAS-TL). Although it is experimentally known that formation of the CS complex influences cysteine production, the exact biological function of the CS complex, the mechanism of reciprocal regulation of the constituent enzymes and the structure of the complex are still poorly understood. Here, we used docking techniques to construct a model of the CS complex from mitochondrial Arabidopsis thaliana. The three-dimensional structures of the enzymes were modeled by comparative techniques. The C-termini of SAT, missing in the template structures but crucial for CS formation, were modeled de novo. Diffusional encounter complexes of SAT and OAS-TL were generated by rigid-body Brownian dynamics simulation. By incorporating experimental constraints during Brownian dynamics simulation, we identified complexes consistent with experiments. Selected encounter complexes were refined by molecular dynamics simulation to generate structures of bound complexes. We found that although a stoichiometric ratio of six OAS-TL dimers to one SAT hexamer in the CS complex is geometrically possible, binding energy calculations suggest that, consistent with experiments, a ratio of only two OAS-TL dimers to one SAT hexamer is more likely. Computational mutagenesis of residues in OAS-TL that are experimentally significant for CS formation hindered the association of the enzymes due to a less-favorable electrostatic binding free energy. Since the enzymes from A. thaliana were expressed in Escherichia coli, the cross-species binding of SAT and OAS-TL from E. coli and A. thaliana was explored. The results showed that reduced cysteine production might be due to a cross-binding of A. thaliana OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation. (C) 2008 Elsevier Ltd. All rights reserved.