Understanding and Harnessing the Structural Features that Govern HMG-CoA Reductase Activity
The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), which catalyzes the reduction of HMG-CoA to mevalonate using two equivalents of the cofactor NAD(P)H, is part of the mevalonate pathway, found in all kingdoms of life. This pathway is at the heart of natural product biosynthesis constituting one of the obligate routes to producing the building blocks for isoprenoids, which represent the largest and most diverse class of natural products. Natural products and its derivatives continue to provide important compounds in the fields of drug development, biomedical engineering, and commercially-driven products. Therefore, HMGR, which performs the rate-limiting step of the mevalonate pathway garners significant attention in these fields. Understanding of the mechanism of HMGR remains incomplete, with gaps pertaining to the role of a putative C-terminal flap domain (CTD) responsible for helping to modulate the positioning of active site residues as well as the flux of substrates, intermediates, and products during the reaction. Moreover, investigation into structural determinants of cofactor specificity, which contribute towards varied cofactor preferences observed among HMGR homologs, point to a cofactor helix as being crucial, and warrants further analysis. Here, class II HMGRs from Delftia acidovorans (DaHMGR) and Streptococcus pneumoniae (SpHMGR) are used as case-studies in elucidating biophysical, kinetic, and X-ray crystallographic features that aid in our understanding of the three-dimensional characteristics of class II HMGR including its catalytic and oligomeric states in solution, the role of the CTD in the mechanism and features governing cofactor specificity. We provide the first crystal structures of DaHMGR alongside complementary kinetic studies showing it to be an NAD-preferring HMGR. We observe that NAD-preferring HMGRs are able to form hexamers whereas NADP-preferring HMGRs predominate as dimers. Structures obtained with the CTD in novel locations expands our knowledge on this domain. We observe a flipped conformation of the CTD that appears to capture an intermediate state. The importance of the cofactor helix is manifest through protein engineering efforts that switch specificity by switching this motif between HMGRs. This work expands on the current class II HMGR paradigm as it relates to structural and functional dynamics, providing greater insight into this biologically important enzyme.
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