Structure of the Escherichia coli malate synthase G:pyruvate:acetyl-coenzyme A abortive ternary complex at 1.95 Å resolution
Structure of the Escherichia coli malate synthase G:pyruvate:acetyl-coenzyme A abortive ternary complex at 1.95 Å resolution
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DOI：
10.1110/ps.03174303
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发表时间：
2003-09-01
期刊：
影响因子：
8
通讯作者：
Remington, SJ
中科院分区：
文献类型：
--
作者：
Anstrom, DM;Kallio, K;Remington, SJ
苹果酸合酶是乙醛酸途径的一种酶，催化乙酰辅酶A（乙酰辅酶A）和乙醛酸的缩合和随后的水解以形成苹果酸和辅酶A。在本研究中，我们提出了1.95埃分辨率的晶体结构的大肠杆菌苹果酸合酶亚型G镁，丙酮酸，乙酰辅酶A的复合物，我们比较它与以前确定的结构的底物和产品复合物。结果揭示了酶如何识别和激活底物乙酰辅酶A，以及与底物结合相关的构象变化，这可能对催化作用很重要。基于这些结果和活性位点残基的诱变，提出Asp 631和Arg 338协同作用以在限速步骤中形成乙酰辅酶A的烯醇阴离子。高度保守的Cys 617，这是直接相邻的推定催化碱天冬氨酸63 - 1，似乎被氧化为半胱氨酸-次磺酸。这可以解释早期观察到的敏感性的酶的失活和聚集后，X-射线照射，并表明，半胱氨酸氧化可能在体内苹果酸合酶活性的氧化还原调节中发挥作用。越来越多的证据表明，乙醛酸途径的酶是几种致病生物的毒力因子，特别是结核分枝杆菌和白色念珠菌。在这项研究中描述的结果增加了洞察的催化机制，并可能是有用的抑制性化合物的设计作为可能的抗菌剂。
Malate synthase, an enzyme of the glyoxylate pathway, catalyzes the condensation and subsequent hydrolysis of acetyl-coenzyme A (acetyl-CoA) and glyoxylate to form malate and CoA. In the present study, we present the 1.95 Angstrom-resolution crystal structure of Escherichia coli malate synthase isoform G in complex with magnesium, pyruvate, and acetyl-CoA, and we compare it with previously determined structures of substrate and product complexes. The results reveal how the enzyme recognizes and activates the substrate acetyl-CoA, as well as conformational changes associated with substrate binding, which may be important for catalysis. On the basis of these results and mutagenesis of active site residues, Asp 631 and Arg 338 are proposed to act in concert to form the enolate anion of acetyl-CoA in the rate-limiting step. The highly conserved Cys 617, which is immediately adjacent to the presumed catalytic base Asp 63 1, appears to be oxidized to cysteine-sulfenic acid. This can explain earlier observations of the susceptibility of the enzyme to inactivation and aggregation upon X-ray irradiation and indicates that cysteine oxidation may play a role in redox regulation of malate synthase activity in vivo. There is mounting evidence that enzymes of the glyoxylate pathway are virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. The results described in this study add insight into the mechanism of catalysis and may be useful for the design of inhibitory compounds as possible antimicrobial agents.