Victor W. Rodwell

Regulation of HMG-CoA Reductase

Publisher Summary This chapter discusses the regulation of HMG-CoA reductase, which catalyzes the rate-limiting reaction of hepatic sterol synthesis. Both mitochondrial and extramitochondrial forms of acetoacetyl-CoA thiolase and the HMG-CoA synthase are present in liver tissue. The reductase activity of cultured mammalian cells or of bacteria is readily assayed in crude homogenates. The HMG-CoA reductase activity may be readily assayed in the post-mitochondrial supernatant fraction obtained by high-speed centrifugation of homogenates of tissues, including liver. The chapter also discusses familial hypercholesterolemia (FH), which is an autosomal, dominant genetic disorder estimated to affect 0.1–0.2% of the population. FH may involve defects in the regulation of both cholesterol synthesis and degradation. The data for adults heterozygous for FH indicate that the rate of cholesterol synthesis in vivo is subnormal. The basic defect in FH may be a low affinity of the cellular plasma membrane for cholesterol. This defect, observed in fibroblasts and leukocytes, leads to an impaired uptake of cholesterol from serum lipoproteins and an enhanced release of cellular cholesterol. If this defect is present in the liver, it could account for the relatively ineffective feedback suppression of cholesterol synthesis.

Micro assay for 3-hdyroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts

Abstract A rapid, highly reproducible, micro-incubation, direct thin-layer chromatographic assay for 3-hydroxy-3-methylglutaryl-CoA reductase is described. The isolation and quantitation of mevalonic acid is simplified by direct application of the deproteinized, acidified incubation mixture to thin-layer chromatography sheets. The tedious and variable extraction of mevalonic acid into ether is eliminated by this procedure. Background levels are lower than those obtained with comparable assays and permit quantitation of as little as 30 pmoles of mevalonic acid. The method is illustrated by analysis of 3-hydroxy-3-methylglutaryl CoA reductase from rat liver, cultured L cells, and solubilized microsomal reductase. The rapid inactivation of reductase by MgATP and the low levels of reductase activity in cholesterol-treated L cells are accurately determined by using the micro-incubation, direct thin-layer chromatography method.

The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases

SummaryThe enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. The enzyme is found in eukaryotes and prokaryotes; and phylogenetic analysis has revealed two classes of HMG-CoA reductase, the Class I enzymes of eukaryotes and some archaea and the Class II enzymes of eubacteria and certain other archaea. Three-dimensional structures of the catalytic domain of HMG-CoA reductases from humans and from the bacterium Pseudomonas mevalonii, in conjunction with site-directed mutagenesis studies, have revealed details of the mechanism of catalysis. The reaction catalyzed by human HMG-CoA reductase is a target for anti-hypercholesterolemic drugs (statins), which are intended to lower cholesterol levels in serum. Eukaryotic forms of the enzyme are anchored to the endoplasmic reticulum, whereas the prokaryotic enzymes are soluble. Probably because of its critical role in cellular cholesterol homeostasis, mammalian HMG-CoA reductase is extensively regulated at the transcriptional, translational, and post-translational levels.

Diurnal variation and cholesterol regulation of hepatic HMG-CoA reductase activity☆

HMG-CoA reductase exhibits a diurnal rhythm. Cycloheximide injection completely prevents both the observed 9.5 fold rise in activity and the loss of activity during daylight. This is compatible with the existence of a specific, labile,degradative or inactivating protein for HMG-CoA reductase. Effects of cholesterol on reductase activity were also studied. KM and Vmax for reductase from normal- and cholesterol-fed rats are 8.1×10−5 M and 0.33 nmole/min/mg and 6.9x10−5 M and 0.0404 nmole/min/mg respectively. Reductase from both sources is substrate-inhibited by HMG-CoA. Mixing experiments suggest that a soluble inhibitor of IM -CoA reductase does not cause the profound drop in activity observed in cholesterol-fed rats.

Thin-layer chromatographic assay for HMG-CoA reductase and mevalonic acid

Abstract An improved TLC system for the isolation and quantitation of mevalonic acid is described. This method has been applied to a variety of biological systems. The identification of the biological product as mevalonic acid has been confirmed by rechromatography with mevalonic acid standards. The procedure is simple, reproducible, and suitable for large numbers of HMG-CoA reductase assays. The system equals available GLC methods in sensitivity and is decidedly faster, particularly if large numbers of analyses are to be performed. Using a purified bacterial HMG-CoA reductase we obtained excellent agreement between a spectral assay and the TLC method presented. Prior extraction of mevalonic acid may be omitted if less exact results will suffice. Although the method is proposed for assay of HMG-CoA reductase, it is applicable to isolation and quantitation of mevalonolactone from a variety of biological systems.

Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.

Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of Staphylococcus aureus

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K(i) of 320 microM, over 10(4) higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases

The biosynthesis of isopentenyl diphosphate (isopentenyl pyrophosphate), the precursor of isoprenoids in all forms of life, occurs by two distinct metabolic pathways, the mevalonate pathway (Fig. 1) and the glyceraldehyde 3-phosphate/pyruvate pathway, often termed the nonmevalonate pathway (17). Whereas many gram-negative bacteria employ the nonmevalonate pathway (26), humans, other eukaryotes, archaea, grampositive cocci, and the spirochete Borrelia burgdorferi utilize the enzymes and intermediates of the mevalonate pathway (15, 16, 20, 21, 26). This review addresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the catalyst for the ratelimiting reaction of the mevalonate pathway for isopentenyl diphosphate biosynthesis. HMG-CoA reductase catalyzes the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate: The reaction proceeds in three stages, the first and third of which are reductive, and it involves successive formation of enzyme-bound mevaldyl-CoA and mevaldehyde. While mevaldehyde is not released during the course of the reaction, HMG-CoA reductase catalyzes two reactions of free mevaldehyde. Reaction 2 resembles the third stage, and reaction 3 resembles the reverse of stages 1 and 2 of the overall reaction 1.

Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis

Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.

In vitro phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: analysis of 32P-labeled, inactivated enzyme.

Abstract Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was inactivated with Mg 2+ and [γ- 32 P]ATP, then solubilized and purified to homogeneity. The 32 P radioactivity was precipitated by antibody to homogeneous rat liver reductase and comigrated with nonprecipitated, homogeneous reductase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturing conditions, 32 P radioactivity comigrated with reductase protein and activity on polyacrylamide gels. These results provide direct support for the concept that the enzyme is covalently phosphorylated during the in vitro incubation of microsomes with Mg 2+ and ATP.