Mentor: Dr.Kung (Chemistry Department)
The mevalonate pathway is an important metabolic pathway for the production of dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), which serve as the basis for the biosynthesis of isoprenoids that are good candidates for diverse biofuel and drug compounds. The rate limiting and first committed step in the mevalonate pathway is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGR). A substrate-induced closure of a C-terminal flap domain of HMGR was found in the Pseudomonas mevalonii and Streptococcus pneumoniae enzymes; this flap domain was shown to be involved in the binding of NADH for the reduction of HMG-CoA to mevalonate. However, the structure of this domain has not been observed so far in NADPH-utilizing yeast or human HMGR because the flexibility of the domain poses a challenge for crystallography. To complete the understanding of HMGR and reveal the structural mechanism of NADPH binding, the residue Ser1019 near the C-terminus of the enzyme will be mutated to a cysteine by site-directed mutagenesis to introduce a disulfide bond that can anchor the domain in place for X-ray crystallography. Comparative analysis of this flap domain in both NADH- and NADPH- binding HMGR could provide insight into difference between the two binding mechanisms; based on this knowledge, the enzyme may be further engineered to switch coenzyme specificity, facilitating various syntheses achieved through the mevalonate pathway.