An Atomic-Level Perspective of HMG-CoA-Reductase: The Target Enzyme to Treat Hypercholesterolemia.

This review provides an updated atomic-level perspective regarding the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR), linking the more recent data on this enzyme with a structure/function interpretation. This enzyme catalyzes one of the most important steps in cholesterol biosynthesis and is regarded as one of the most important drug targets in the treatment of hypercholesterolemia. Taking this into consideration, we review in the present article several aspects of this enzyme, including its structure and biochemistry, its catalytic mechanism and different reported and proposed approaches for inhibiting this enzyme, including the commercially available statins or the possibility of using dimerization inhibitors.

New Crystallographic Snapshots of Large Domain Movements in Bacterial 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the first committed step of the mevalonate pathway, which is used across biology in the biosynthesis of countless metabolites. HMGR consumes 2 equiv of the cofactor NAD(P)H to perform the four-electron reduction of HMG-CoA to mevalonate toward the production of steroids and isoprenoids, the largest class of natural products. Recent structural data have shown that HMGR contains a highly mobile C-terminal domain (CTD) that is believed to adopt many different conformations to permit binding and dissociation of the substrate, cofactors, and products at specific points during the reaction cycle. Here, we have characterized the HMGR from Delftia acidovorans as an NADH-specific enzyme and determined crystal structures of the enzyme in unbound, mevalonate-bound, and NADH- and citrate-bound states. Together, these structures depict ligand binding in both the active site and the cofactor-binding site while illustrating how a conserved helical motif confers NAD(P)H cofactor specificity. Unexpectedly, the NADH-bound structure also reveals a new conformation of the CTD, in which the domain has "flipped" upside-down, while directly binding the cofactor. By capturing these structural snapshots, this work not only expands the known range of HMGR domain movement but also provides valuable insight into the catalytic mechanism of this biologically important enzyme.

Metabolic control of YAP and TAZ by the mevalonate pathway.

The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases.

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD(+)-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.