Isoprenoids represent the largest and most structurally diverse class of natural products. Used as drugs for the treatment of a multitude of human diseases, isoprenoids and their derivatives can now be produced in microorganisms expressing the enzymes of the mevalonate pathway, which is responsible for the biosynthesis of isoprenoid precursors. The enzyme targets of in vivo mevalonate pathway regulation are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) and mevalonate kinase (MK). For both of these enzymes, key structural and mechanistic information is currently lacking, hindering the further development of microbial systems for isoprenoid production. Specifically, the structural basis of HMGR cofactor specificity for either NADH or NADPH, which differs among HMGR homologs, is as yet unknown. For MK, which is subject to tight feedback inhibition, the identity of the inhibitors and their inhibition potencies vary widely across MKs frm different organisms, yet the structural underpinnings that govern such a variance in inhibition profiles are entirely unexplored. In this study, these gaps in fundamental structural knowledge will be addressed directly. Using X-ray crystallography in conjunction with complementary biochemical studies, the structural bases of both HMGR cofactor specificity and MK inhibition variance among enzyme homologs will be investigated. In addition, to further probe the enzyme structure-function relationship, chimeric enzymes will be constructed and studied in order to alter or switch the differential activities between enzymes from different organisms. Therefore, the proposed work will not only reveal the structural determinants to critical HMGR and MK properties that remain unstudied, it will also employ structure-guided protein modification to yield new enzymes with more desirable cofactor preferences and inhibitory responses that can be used in the microbial production of isoprenoid natural products.

Public Health Relevance

Isoprenoids are the largest and most structurally diverse class of natural products and include compounds currently used as drugs to treat a wide variety of diseases ranging from cancer to malaria, in addition to promising new drug candidates. The biological mass production of isoprenoids in microorganisms is now possible by expressing enzymes of the mevalonate pathway, which is responsible for the biosynthesis of isoprenoid precursors. Through structural and biochemical study of the activity and inhibition of the key regulatory enzymes of the mevalonate pathway, the goal of this work is to obtain detailed mechanistic and structural insight into enzyme catalysis and regulation, and thus to enable the creation of modified enzymes endowed with more desirable functions for tailored isoprenoid drug biosynthesis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Macromolecular Structure and Function A Study Section (MSFA)
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Barski, Oleg
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Bryn Mawr College
Schools of Arts and Sciences
Bryn Mawr
United States
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