Structural investigation and manipulation of key regulatory enzymes in the mevalonate pathway of isoprenoid biosynthesis

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PROJECT SUMMARY Isoprenoids comprise the largest and most structurally diverse class of natural products. Used as drugs in the treatment of many human diseases, isoprenoids and their derivatives are now being produced in microorganisms engineered to express the enzymes of the mevalonate pathway, which is responsible for the biosynthesis of the universal isoprenoid precursors. The key enzymes of the mevalonate pathway are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), which catalyzes the committed and rate-limiting step, and mevalonate kinase (MK), which is a primary target for regulation of the pathway. For both of these enzymes, critical structural and mechanistic information is lacking, hindering the further development of biological systems for isoprenoid production. In particular, the structural basis of HMGR cofactor specificity for either NADH or NADPH, which differs among HMGR homologs in biology, remains unclear (Specific Aim 1). Further, a C-terminal domain of HMGR has recently been visualized in many different conformations, yet the purpose of this domain in HMGR function and catalysis is a mystery (Specific Aim 2). For MK, key features of the catalytic mechanism remain unknown due to a dearth of structural information on substrate and ligand binding (Specific Aim 3). In addition, MK is subject to feedback inhibition, where the identities and potencies of inhibitors vary widely among MK homologs, yet the structural basis governing this variance in inhibition profiles is unexplored (Specific Aim 4). In the proposed work, these gaps in fundamental structural and biochemical knowledge will be addressed using X-ray crystallography in conjunction with complementary biochemical, biophysical, and computational studies. In addition, to further probe the enzyme structure-function relationship, mutant, modified, and chimeric enzymes will be created to study the varying catalytic activities and ligand-binding features of enzymes from different organisms. Therefore, this work will not only uncover the structural determinants to key HMGR and MK functions, it will also use structure-guided protein modification to construct new enzymes with altered cofactor specificities, ligand-binding features, catalytic activities, and inhibitory responses that may be used in the biological production of isoprenoids and their derivatives. Further, by gaining deeper insight into the structural and functional differences between HMGR and MK homologs, this work may also lead to the development of new antimicrobial drugs that target mevalonate pathway enzymes in human pathogens.