The 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.
Gene organization and evolutionary history
The human hmgr gene that encodes the single human HMG-CoA reductase is located on chromosome 5, map location 5q13.3-5q14, and is over 24.8 kilobases (kb) long. The 20 exons of the 4,475-nucleotide transcript, which range in size from 27 to 1,813 base-pairs, encode the membrane-anchor domain (exons 2-10), a flexible linker region (exons 10 and 11), and the catalytic domain (exons 11-20) of the resulting 888-residue polypeptide (Figure 1).
Figure 1. Schematic representation of the human hmgr gene and the human HMGRH and P. mevalonii HMGRP proteins. (a) The exon-intron structure of the human hmgr gene, which extends from position 74717172 to position 74741998 of the human genome; positions refer to the Ensembl Transcript ID for the human hmgr gene (ENST00000287936 ). The numbers indicate the start and end of each exon and intron and refer to the position in the human genome sequence, omitting the first three digits (747); exons are indicated as filled boxes. Exon 1 is an untranslated region (UTR), as are the last 1,758 nucleotides of exon 20. The exons encoding the membrane-anchor domain, a flexible linker region, and the catalytic domain are indicated below the gene structure. (b) Human HMGR protein (HMGRH) is comprised of three domains: the membrane anchor domain, a linker domain, and a catalytic domain; within the catalytic domain subdomains have been defined. The N domain connects the L domain to the linker domain; the L domain contains an HMG-CoA binding region; and the S domain functions to bind NADP(H). The cis-loop (indicated by an asterisk), a region present only in HMGRH but not HMGRP, connects the HMG-CoA-binding region with the NADPH-binding region. (c) The HMGRP protein does not contain the membrane-anchor domain or the linker domain but has a catalytic domain containing a large domain with an HMG-CoA binding region, and a small, NAD(H)-binding domain. The active site of HMG-CoA reductase is present at the homodimer interface between one monomer that binds the nicotinamide dinucleotide and a second monomer that binds HMG-CoA. The numbers underneath the diagrams in (b,c) denote amino acids (in the single-letter amino-acid code) that are implicated in catalysis; S872 of HMGRH is reversibly phosphorylated. At the extreme carboxyl terminus of each enzyme is a flap domain (approximately 50 amino acids in HMGRP and 25-30 amino acids in HMGRH) that closes over the active site during catalysis; the flap domain is indicated by shading in (b,c).
Genome sequencing has identified hmgr genes in organisms from all three domains of life, and over 150 HMGR sequences are recorded in public databases. Higher animals, archaea, and eubacteria have only a single hmgr gene, although the lobster has both a soluble and a membrane-associated isozyme, both of which are encoded by a single gene). By contrast, plants, which use both HMGR-dependent and HMGR-independent pathways to synthesize isoprenoids, have multiple HMGR isozymes that appear to have arisen by gene duplication and subsequent sequence divergence . Yeast has two HMGR isozymes derived from two different genes (hmgr-1 and hmgr-2). Comparison of amino-acid sequences and phylogenetic analysis reveals two classes of HMGR, the Class I enzymes of eukaryotes and some archaea and the Class II enzymes of certain eubacteria and archaea, suggesting evolutionary divergence between the two classes (Figure 2, Table 1) [2,3]. The catalytic domain is highly conserved in eukaryotes, but the membrane-anchor domain (consisting of between two and eight membrane-spanning helices) is poorly conserved, and the HMGRs of archaea and of certain eubacteria lack a membrane-anchor domain.
Figure 2. A phylogenetic tree of HMGRs. The tree includes 98 selected organisms that have hmgr genes; for plants, which have multiple isoforms, only isoform 1 of each species is included in the tree. Roman numerals indicate the division of the family into two classes [2,3]. Phylogenetic analysis was performed using aligned amino-acid sequences of HMGR catalytic domains; membrane anchor domains were excluded from analysis. Amino-acid sequence alignments were generated using ClustalW  and the phylogenetic tree constructed with TreeTop  using the cluster algorithm with PHYLIP tree-type output. Full species names and GenBank accession numbers of the sequences used are provided in Table 1.
Table 1. Details of the sequences used for the phylogenetic tree in Figure 2
Characteristic structural features
The HMGRs of different organisms are multimers of a species-specific number of identical monomers. High-resolution crystal structures have been solved for the Class I human enzyme (HMGRH) [4,5] and for the Class II enzyme of Pseudomonas mevalonii (HMGRP) [6,7], including protein forms bound to either the HMG-CoA substrate or the coenzyme (NADH or NADPH) or both, or bound to statin drugs, which are potent competitive inhibitors of HMGR activity and thus lower cholesterol levels in the blood [8,9]. As reviewed in detail by Istvan , structural comparisons reveal both similarities and significant differences between the two classes of enzyme. The human HMGR has three major domains (catalytic, linker and anchor), whereas the P. mevalonii HMGR has only the catalytic domain (Figure 1).
Both HMGRH and HMGRP have a dimeric active site with residues contributed by each monomer, and a non-Rossman-type coenzyme-binding site (a three-dimensional structural fold that contains a nucleotide-binding motif and is found in many enzymes that use the dinucleotides NADH and NADPH for catalysis). The core regions containing the catalytic domains of the two enzymes have similar folds. Despite differences in amino-acid sequence and overall architecture, functionally similar residues participate in the binding of coenzyme A by the two enzymes, and the position and orientation of four key catalytic residues (glutamate, lysine, aspartate and histidine) is conserved in both classes of HMGR.
Unlike the central cores, the amino- and carboxy-terminal regions of the catalytic domains show little similarity between the human and P. mevalonii HMGR structures. The active site of HMG-CoA reductase is at the interface of the homodimer between one monomer that binds the nicotinamide dinucleotide and a second monomer that binds the HMG-CoA. In human HMGR, the catalytic lysine is found on the monomer that binds the HMG-CoA and comes from the so-called cis-loop (a section that connects the HMG-CoA-binding region with the NADPH-binding region). In contrast, the P. mevalonii HMGR lacks the cis-loop and the catalytic lysine is contributed by the monomer that binds the nicotinamide dinucleotide. HMGRP crystallizes as a trimer of dimers (which are composed of identical subunits), but HMGRH crystallizes as a tetramer (of identical units). HMGRP uses NADH as a coenzyme, whereas HMGRH uses NADPH, but mutation to alanine of the aspartyl residue of HMGRP that normally blocks binding of NADPH can allow NADPH to serve - albeit poorly - as the coenzyme for HMGRP. A 180° difference in the orientation of the nicotinamide ring of the coenzyme suggests that that the stereospecificity of the HMGRH hydrogen transfer is opposite to that of HMGRP.
Comparisons between the HMGRP and HMGRH structures reveal an overall similarity in how they bind statins, which inhibit activity by blocking access of HMG-CoA to the active site. There is a considerable difference in specific interactions with inhibitor between the two enzymes, however [8,9], accounting for the almost 104-fold higher Ki values for inhibition of HMGRP by statin relative to the inhibition of HMGRH (Ki is the equilibrium constant for an inhibitor binding to an enzyme). There are significant differences in the regions of the two proteins that bind statins. In both enzymes the portion of the statin that resembles HMG (see Figure 3) occupies the HMG portion of the HMG-CoA-binding pocket, and the non-polar region partially occupies a portion of the coenzyme-A-binding site. For HMGRP, this impairs closure over the active site of the 'tail' domain that contains the catalytic histidine.
Figure 3. Structures of lovastatin, a statin drug that competitively inhibits HMGR, and of HMG-CoA. It can be seen that the portion of the drug shown here at the top resembles the HMG portion of HMG-CoA.
Localization and function
HMGRs of eukaryotes are localized to the endoplasmic reticulum (ER), and are directed there by a short portion of the amino-terminal domain (prokaryotic HMGRs are soluble and cytoplasmic). In humans, the reaction catalyzed by HMGR is the rate-limiting step in the synthesis of cholesterol, which maintains membrane fluidity and serves as a precursor for steroid hormones. In plants, a cytosolic HMG-CoA reductase participates in the synthesis of sterols, which are involved in plant development, certain sesquiterpenes, which are important in plant defense mechanisms against herbivores, and ubiquinone, which is critical for cellular protein turnover. In plastids, however, these compounds are synthesized via a pathway that does not involve mevalonate or HMGR . Various plant HMGR isozymes function in fruit ripening and in the response to environmental challenges such as attack by pathogens. In yeast, either of the two ER-anchored HMGR isozymes can provide the mevalonate needed for growth.
The reaction catalyzed by HMGR is:
(S)-HMG-CoA + 2 NADPH + 2 H+ → (R)-mevalonate + 2 NADP+ + CoA-SH.
with the (S)-HMG-CoA and (R)-mevalonate designations referring to the stereochemistry of the substrate and product (enzymatic reactions are stereospecific and the (R)-HMG-CoA isomer is not a substrate for HMGR). This three-stage reaction involves two reductive stages and the formation of enzyme-bound mevaldyl-CoA and mevaldehyde as probable intermediates:
Stage 1: HMG-CoA + NADPH + H+ → [Mevaldyl-CoA] + NADP+
Stage 2: [Mevaldyl-CoA] → [Mevaldehyde] + CoA-SH
Stage 3: [Mevaldehyde] + NADPH + H+ → Mevalonate + NADP+
Kinetic analysis of point mutants of HMGRP and of HMGRH, and inspection of the crystal structures of HMGRP and HMGRH, has identified an aspartate, a glutamate, a histidine, and a lysine that are likely to be important and have suggested their probable roles in catalysis (Figure 4) .
Figure 4. Proposed reaction mechanism for HMGRP [7,18]. The side groups of the key catalytic residues, Lys267, Asp283, Glu83, and His381, are shown, and the substrate and products are shown with R representing the HMG portion. The reaction follows three stages (see text for details). A basically similar mechanism has been proposed for HMGRH .
A highly regulated enzyme, HMGRH is subject to transcriptional, translational, and post-translational control  that can result in changes of over 200-fold in intracellular levels of the enzyme. The transcription factor sterol regulatory element-binding protein 2 (SREBP-2) participates in regulating levels of HMGRH mRNA in response to the level of sterols ; the regulatory process is as follows. At the ER membrane or the nuclear envelope, SREBP-2 binds to SREBP cleavage activating protein (SCAP) to form a SCAP-SREBP complex that functions as a sterol sensor. The proteins Insig-1 and Insig-2 bind to SCAP when cellular cholesterol levels are high and prevent movement of the SCAP-SREBP complex from the ER to the Golgi. In cells depleted of cholesterol, Insig-1 and Insig-2 allow activation of the SCAP-SREBP complex and its translocation to the Golgi, where SREBP is cleaved at two sites. Cleavage releases the amino-terminal basic helix-loop-helix (bHLH) domain, which enters the nucleus, where it functions as a transcription factor that recognizes non-palindromic decanucleotide sequences of DNA called sterol-regulatory elements (SREs). Binding of the bHLH domain of SREBP to an SRE promotes transcription of the hmgr gene.
Degradation of HMGRH involves its transmembrane regions : removal of two or more transmembrane regions abolishes the acceleration of HMGRH degradation that occurs under certain conditions [12,15]: degradation is induced by a non-sterol, mevalonate-derived metabolite alone or by a sterol plus a mevalonate-derived non-sterol metabolite, possibly farnesyl pyrophosphate or farnesol. Four conserved phenylalanines in the sixth membrane span of the transmembrane region are essential for degradation of HMGRH . Insig-1 also functions in the degradation of HMGRH : when cholesterol levels are high, SCAP and HMGRH compete for binding to Insig-1. If SCAP binds Insig-1, the SCAP-Insig-1 complex is retained in the Golgi, whereas if HMGRH binds Insig-1, HMGRH is ubiquinated on lysine 248 and is rapidly degraded through a ubiquitin-proteasome mechanism .
The catalytic activity of the HMGRs of higher eukaryotes is attenuated by phosphorylation of a single serine, which in the case of HMGRH is at position 872 . The location of this serine - six residues from the catalytic histidine, a spacing conserved in all higher eukaryote HMGRs - suggests that the phosphoserine may interfere with the ability of this histidine to protonate the inhibitory CoAS- thioanion that is released in stage 2 of the reaction mechanism. Alternatively, it may interfere with closure of the flap domain, a carboxy-terminal region that is thought to close over the active site to facilitate catalysis, a step thought to be essential for formation of the active site . Subsequent dephosphorylation restores full catalytic activity. HMGR kinase (also called AMP kinase) phosphorylates HMGR; the primary phosphatase in vivo is thought to be protein phosphatase 2A (PP2A), but both phosphatases 2A and 2B can catalyze dephosphorylation of vertebrate HMGR in vitro . HMGRH activity therefore responds to hormonal control through AMP levels and PP2A activity. Phosphorylation of serine 577 of A. thaliana HMGR isozyme 1 by a plant HMGR kinase that does not require 5'-AMP attenuates activity, and restoration of HMGR activity follows from dephosphorylation . As many plant genes encode a putative target serine surrounded by an apparent AMP kinase recognition motif, it is probable that most plant HMGRs are regulated by phosphorylation. Yeast HMGR activity is, however, unaffected by AMP kinase. The phosphorylation state of HMGR does not affect the rate at which the protein is degraded.
Several basic unresolved questions concern how phosphorylation controls the catalytic activity of HMGRs; solution of the structures of phosphorylated HMGRs should reveal more of the precise mechanism. The protein kinases, phosphatases, and signal-transduction pathways that participate in short-term regulation of HMGR activity are yet to be elucidated. Finally, the physiological roles served by the multiple ways in which HMGR is regulated require clarification. On the medical side, continuing intense competition between drug companies for a share of the lucrative worldwide market for hypercholesterolemic agents should result in new statin drugs with modified pharmacodynamic and metabolic properties that not only lower plasma cholesterol levels more effectively but more importantly minimize undesirable side effects.
Proc Natl Acad Sci USA 2003, 100:6866-6871.
A study of the regulation of both mevalonate and mevalonate independent pathways for isoprenoid synthesis in plants.PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mol Genet Metab 1999, 66:122-127.
This article reported the classification of HMG-CoA reductases into Class I and Class II enzymes on the basis of sequence comparison. The authors utilized phylogenetic analysis to analyze a plethora of genomic sequences of various organisms.PubMed Abstract | Publisher Full Text
J Bacteriol 2004, 186:1927-1932.
A review article detailing current research and thought concerning Class II forms of the enzyme, including the HMGRs of many pathogenic bacteria.PubMed Abstract | Publisher Full Text | PubMed Central Full Text
EMBO J 2000, 19:819-830.
This article and  reported the crystal structure of the human HMG-CoA reductase catalytic domain, providing numerous insights into catalysis by a Class I HMG-CoA reductase.PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Biochim Biophys Acta 2000, 1529:9-18.
See PubMed Abstract | Publisher Full Text
Science 1995, 268:1758-1762.
This article reports the first HMG-CoA reductase structure that was solved.PubMed Abstract
Tabernero LD, Bochar DA, Rodwell VW, Stauffacher CV: Substrate-induced closure of the flap domain in the ternary complex structures provides new insights into the mechanism of catalysis by 3-hydroxy-3-methylglutaryl-CoA reductase.
Proc Natl Acad Sci USA 1999, 96:7167-7171.
The original structure of P. mevalonii HMG-CoA reductase  lacked a portion of the enzyme known to be critical for catalysis. This article provided insight into the catalytic mechanism by solving the structure of the original missing region.PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Science 2001, 292:1160-1164.
This article reports a structural explanation for inhibition of human HMG-CoA reductase by statins, which are widely prescribed drugs for hypercholesterolemia.PubMed Abstract | Publisher Full Text
J Biol Chem 2003, 278:19933-19938.
The authors detail the interaction of P. mevalonii HMG-CoA reductase, a Class II enzyme, with statins.PubMed Abstract | Publisher Full Text
Curr Opin Struct Biol 2001, 11:746-751.
A review that provides insight into the relationships between Class I and Class II HMG-CoA reductases, both in terms of structure and evolution.PubMed Abstract | Publisher Full Text
Bochar DA, Friesen JA, Stauffacher CV, Rodwell VW: Biosynthesis of mevalonic acid from acetyl-CoA. In Isoprenoids Including Carotenoids and Steroids. Edited by Cane D. New York: Pergamon Press; 1999::15-44.
A comprehensive review article detailing the catalysis, structure, and regulation of HMG-CoA reductase. It is written from the point of view of natural products synthesis.
Nature 1990, 343:425-430.
The first major report on the regulation of HMG-CoA reductase.PubMed Abstract | Publisher Full Text
J Clin Invest 2002, 109:1125-1131.
A recent review detailing the role of sterol regulatory element binding proteins (SREBPs) in the regulation of cholesterol biosynthesis. This is the transcriptional control for HMG-CoA reductase.PubMed Abstract | Publisher Full Text | PubMed Central Full Text
A classical review article summarizing the role of the membrane anchor domain in HMG-CoA reductase degradation.
Jingami H, Brown MS, Goldstein JL, Anderson RJ, Luskey KL: Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.
J Cell Biol 1987, 104:1693-1704.
The original report of the sterol-mediated regulation of HMG-CoA reductase degradation and localization of the region responsible for mediating this degradation.PubMed Abstract | Publisher Full Text
Xu L, Simoni RD: The inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by sterol regulatory element binding protein cleavage-activating protein requires four phenylalanine residues in span 6 of HMG-CoA reductase transmembrane domain.
Arch Biochem Biophys 2003, 414:232-243.
A study of the structure-function relationships between HMG-CoA reductase degradation and the sterol cleavage activating protein (SCAP).PubMed Abstract | Publisher Full Text
Mol Cell 2003, 11:25-33.
The authors identified the role of the protein insig-1 in regulation of HMG-CoA reductase by degradation.PubMed Abstract | Publisher Full Text
Sever N, Song BL, Yabe D, Goldstein JL, Brown MS, DeBose-Boyd RA: Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.
J Biol Chem 2003, 278:52479-52490.
This study described the relationship between ubiquitination, degradation, and the protein insig-1 in HMG-CoA reductase degradation.PubMed Abstract | Publisher Full Text
Sato R, Goldstein JL, Brown MS: Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion.
Proc Natl Acad Sci USA 1993, 90:9261-9265.
In this study, the authors identified the specific amino acid of mammalian HMG-CoA reductase that is phosphorylated and mediates regulation of HMG-CoA reductase by reversible phosphorylation.PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Endocrinology 2003, 144:5179-5183.
A review article describing the AMP-activated protein kinase (AMPK) that phosphorylates HMG-CoA reductase.PubMed Abstract | Publisher Full Text
Dale S, Arro M, Becerra B, Morrice NG, Boronat A, Hardie DG, Ferrer A: Bacterial expression of the catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase (isoform hmgr1) from Arabidopsis thaliana, and its inactivation by phosphorylation at Ser577 by Brassica oleracea 3-hydroxy-3-methylglutaryl-CoA reductase kinase.
Eur J Biochem 1995, 233:506-513.
A study that illustrated that plant HMG-CoA reductases are probably regulated by reversible phosphorylation.PubMed Abstract | Publisher Full Text
Ensembl information about the human HMG-CoA reductase gene and transcript details.
Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res 1994, 22:4673-4680.
An article describing the CLUSTAL W program, which is used for multiple sequence alignments of amino-acid sequences.PubMed Abstract | PubMed Central Full Text
TreeTop - Phylogenetic tree prediction [http://www.genebee.msu.su/services/phtree_reduced.html] webcite
A program for phylogenetic tree generation.
The NCBI contains a vast amount of sequence information, including protein and nucleic acid sequences for HMG-CoA reductases and information on the sequencing of genomes of organisms containing HMG-CoA reductase isoforms.