Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds
Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds
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DOI：
10.1074/jbc.m507996200
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发表时间：
2006-03-17
影响因子：
4.8
通讯作者：
Hunt, JF
中科院分区：
文献类型：
--
作者：
Forouhar, F;Hussain, M;Hunt, JF
3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）裂解酶催化酮体生成和亮氨酸降解的末端步骤。这种酶的突变会导致一种称为原发性代谢性酸尿症的人类常染色体隐性遗传疾病，这种疾病通常会因无法耐受低血糖而导致患者死亡。在这里，我们提出的HMG-CoA裂解酶从枯草芽孢杆菌和布鲁氏菌的晶体结构，分别在2.7和2.3埃的分辨率。这些酶与人类直向同源物具有大于45%的序列同一性。虽然该酶具有预期的磷酸丙糖异构酶（TIM）桶折叠，催化中心包含一个二价阳离子结合位点，该位点由一簇不变的残基形成，这些残基覆盖桶的核心，与同源模型的预测相反。令人惊讶的是，形成该阳离子结合位点的残基和它们的大部分相互作用伴侣与其他三种TIM桶酶共享，所述其他三种TIM桶酶催化据信通过烯醇化中间体进行的不同的碳-碳键裂解反应（4-羟基-2-酮戊酸醛缩酶、2-异丙基苹果酸合酶和转羧酶5S）。我们建议这个新发现的酶家族命名为“DRE-TIM金属解酶”，该酶家族可能采用涉及不变Asp-Arg-Glu（DRE）三联体的常见催化反应机制。Asp连接二价阳离子，而Arg可能稳定烯醇化物中间体中的电荷积累，Glu保持Asp和Arg的精确结构对齐。我们基于对先前报道的其它DRE-TIM金属解酶的产物复合物的检查和使用人HMG-CoA裂解酶的晶体结构进行的诱导拟合底物对接研究（Fu，et al.（2006）J.Biol.Chem.281，7526-7532的随附论文中报道），提出了HMG-CoA裂解酶的催化反应机制的详细模型。我们的模型与广泛的突变结果一致，并且可以指导后续研究，旨在明确实验阐明该酶的反应机制。
The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 angstrom resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzyme's reaction mechanism.