Exploring the Mechanism of HMG-CoA Reductase With Time-Resolved Laue Diffraction

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The goal of this project is a multifaceted investigation of the molecular mechanism of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from the bacterium Pseudomonas mevalonii. This enzyme catalyzes the interconversion of HMG-CoA and mevalonate, a reaction at the root of the pathway leading to synthesis of active isoprenoids, including such biologically important molecules as steroid hormones, farnesyl pyrophosphate and cholesterol, as well as components of signal transduction networks. Using the structures of the two type members of this enzyme family, the human (Class I) and P. mevalonii (Class II) HMG-CoA reductases in binary and non-productive ternary complexes, a catalytic mechanism has been proposed that can now be investigated in molecular detail. With the discovery of a slow substrate for this enzyme, one can produce crystalline complexes that can provide a series of snapshots of trapped intermediates along the reaction pathway. Initial experiments with these complexes have shown that the P. mevalonii reductase is active in the crystal, which opens the possibility of observing the structural changes during catalysis using time-resolved Laue diffraction. This project will use these approaches to study the underlying structural principles of the mechanism of HMG-CoA reductase. The project will combine crystallographic and kinetic techniques to investigate the mechanistic puzzles of this multistep enzyme reaction.Broader Impacts: The research efforts will be coordinated with educational outreach to undergraduate and high school students. The project provides an outstanding opportunity for training of undergraduate students. In addition, there will be a strong involvement in a collaborative research effort with local high schools. As Director of the ISEF accredited Regional Science and Engineering Fair, the P.I. works closely with three local high school teachers who teach an advanced research class. These collaborative high school research programs teach the basics of science and scientific research at the high school, and then match students with researchers at Purdue for more advanced studies. The P.I. works closely with these teachers to look for appropriate laboratory matches, has hosted high school researchers in the laboratory and accompanies the teachers and student winners to the state and international fairs. These activities will continue in the future.