MOLECULAR MECHANISM OF HMG-COA REDUCTASE

我们的实验室正在进行一个项目， 酶3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）的作用机制 还原酶的X射线晶体学方法。 HMG-CoA还原酶催化 HMG-CoA和甲羟戊酸的相互转化，这是 导致类异戊二烯脂质合成的途径，包括 胆固醇及其衍生物。 在哺乳动物中， HMG-CoA还原酶是胆固醇生物合成的关键步骤，因此HMG-CoA还原酶是 被认为是控制胆固醇产生的主要目标， vivo. 我们已经解决了假单胞菌的2.8埃结构 mevalonii酶，哺乳动物的催化和结构模型 酵素 这种细菌HMG-CoA还原酶的结构揭示了 紧密结合的二聚体，具有明确的活性位点裂缝，位于 二聚体界面 初步研究表明，晶体酶- 可以产生底物复合物，其指示参与 底物的结合和酶的催化活性。 在本研究中，我们建议扩大我们的研究HMG-CoA还原酶 对于天然酶和酶底物的更高分辨率 复合物，开始分子解剖的分子与网站- 定向诱变，并使用这种细菌酶结构作为 研究哺乳动物HMG-CoA还原酶及其相互作用的模型 服用抗胆固醇药物 我们的具体目标是：（1）扩大 电流结构到2.4埃分辨率，2）产生二进制 这种酶的所有底物的复合物和稳定的非生产性 三元络合物，3）收集关于这些络合物的高分辨率数据 并完善它们的结构，以研究 分子机制; 4）用晶体学方法研究 这些酶在关键残基中含有突变体， 配合物 为了研究哺乳动物的系统，我们将模拟 哺乳动物HMG-CoA还原酶的结构对 细菌酶和6）研究抗胆固醇药物的结合， 首先是P. mevalonii还原酶， 最小化，到模型哺乳动物结构。 最后，我们将扩展 这些结构研究通过7）试图结晶催化剂 结构域的叙利亚仓鼠HMG-CoA还原酶，8）通过调查 解决这个C-末端区域的结构的可能性 通过NMR研究和9）通过开始结晶试验， 与HMG-CoA代谢相关的其他酶、HMG-CoA合酶和HMG-CoA 裂合酶

Our laboratory is pursuing a project to understand the molecular mechanism of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by X-ray crystallographic methods. HMG-CoA reductase catalyzes the interconversion of HMG-CoA and mevalonate, a reaction at the root of the pathway leading to synthesis of isoprenoid lipids, including cholesterol and its derivatives. In mammals this reaction is the first committed step in cholesterol biosynthesis, and so HMG-CoA reductase is considered a primary target for the control of cholesterol production in vivo. We have solved the 2.8 angstroms structure of the Pseudomonas mevalonii enzyme, a catalytic and structural model for the mammalian enzyme. The structure of this bacterial HMG-CoA reductase reveals a tightly bound dimer with a well defined active site cleft located at the dimer interface. Preliminary studies have shown that crystalline enzyme- substrate complexes can be produced which indicate residues involved in the binding of the substrates and the catalytic activity of the enzyme. In this research we propose to extend our studies of HMG-CoA reductase to higher resolution for both the native enzyme and the enzyme-substrate complexes, to begin a molecular dissection of the molecule with site- directed mutagenesis and to use this bacterial enzyme structure as a model to investigate the mammalian HMG-CoA reductase and its interactions with anticholesterol drugs. Our specific aims are 1) to extend the current structure to 2.4 angstroms resolution, 2) to produce binary complexes of all the substrates of this enzyme and stable nonproductive ternary complexes, 3) to collect high resolution data on these complexes and refine their structures in order to investigate the details of the molecular mechanism and 4) to study by crystallographic methods the enzymes containing mutants in critical residues suggested by these complexes. In order to study the mammalian system we will 5) model the structure of the mammalian HMG-CoA reductase on the structure of the bacterial enzyme and 6) investigate the binding of anticholesterol drugs, first to the P. mevalonii reductase and the, using techniques of energy minimization, to the model mammalian structure. Finally we will extend these structural studies by 7) attempting to crystallize the catalytic domain of the Syrian hamster HMG-CoA reductase, 8) by investigating the possibility of solving the structure of the C-terminal region of this molecule by an NMR study and 9) by beginning crystallization trials on other enzymes related to HMG-CoA metabolism, HMG-CoA synthase and HMG-CoA lyase.