Eri Arai

Advisor: Dr. Yan Kung

Department of Chemistry

 

Computational and Experimental Studies of the Molecular Basis of Coenzyme Specificity in HMG-CoA Reductase

 

The mevalonate (MEV) pathway is an important metabolic pathway responsible for the conversion of acetyl-CoA into isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are precursors of isoprenoid natural products and steroids. The first committed and rate-limiting step of the MEV pathway is the reduction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, a reaction catalyzed by HMG-CoA reductase (HMGR). HMGR plays a key role in the production of cholesterol in the body and is the sole target enzyme in widely available cholesterol-lowering statin drugs. To date, the structural basis of cofactor specificity in NADH-utilizing class II HMGR, such as Pseudomonas mevalonii and Streptococcus pneumoniae enzymes, has been hypothesized to involve a C-terminal domain that acts as a flexible flap for NADH binding. However, the structural basis of cofactor specificity in NADPH-utilizing class I HMGR, present in human and yeast, remains unknown. Comparative analysis between the C-terminal domains of both NADH- and NADPH- binding HMGR will be studied using computational modeling programs to gain better molecular understanding of HMGR cofactor recognition and specificity. Based on this knowledge, the enzymes will be further engineered using site-directed mutagenesis at the C-terminal domain to disrupt the interactions in its vicinity. The ability of the enzymes to use NADH and NADPH will be monitored in vitro using UV-vis absorbance spectroscopy. Understanding the molecular basis of HMGR cofactor specificity will enable the construction of modified enzymes that can be used for more efficient microbial production of isoprenoid drug compounds.