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.
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