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. (Fig.1)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, gram-positive 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 rate-limiting 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.
HMG-CoA reductase also catalyzes the reverse of reaction 1, the oxidative acylation of (R)-mevalonate to (S)-HMG-CoA (reaction 4).
This enzyme is one of a few four-electron oxidoreductases. Two moles of reduced pyridine nucleotide coenzyme is oxidized during the reduction of 1 mol of the thioester group of HMG-CoA to the primary hydroxyl group of mevalonate.
Site-directed mutagenesis of HMG-CoA reductase has implicated a histidine (7, 8), an aspartate (9), and a glutamate (25), residues that are conserved in all forms of the enzyme, as critical for catalysis. An active-site lysine detected in the first crystal structure of this enzyme (18) was confirmed as a fourth critical residue by mutagenesis (4, 5) and by inspection of the crystal structures of ternary complexes (13, 23). The proposed role of the histidine is to protonate the departing CoA thioanion. If retained, this thioanion would attack bound mevaldehyde and block completion of the overall reaction (9). The catalytic lysine, aspartate, and glutamate form a hydrogen bond-linked network that interacts with the carbonyl group of HMG-CoA. The active-site aspartate participates in both reductive stages of the overall reaction, is central to the hydrogen bond network, and may be part of a proton shuttle (9). The active-site glutamate participates in the second reductive stage of the reaction (9, 25), and the active-site lysine appears to stabilize the mevaldyl-CoA intermediate (23) (Fig. (Fig.22).
Inspection of primary-structure alignments of representative HMG-CoA reductases from eukaryotes, archaea, and bacteria led Bochar et al. (3) to distinguish two distinct classes of HMG-CoA reductases. The distinction between classes rested initially on the observation that the number of conserved residues and sequences was significantly higher within a single class of HMG-CoA reductases than across the two classes and on the different location of the active-site lysine (Fig. (Fig.3).3). Subsequent comparison of the crystal structures of Pseudomonas mevalonii and human HMG-CoA reductases established that this lysine, which is conserved only within each class of the enzyme, was present on a different structural element in the two classes (13).
The primary sequence differences between the two classes parallel the evolutionary diversity of the organisms that harbor enzymes belonging to each class (26). Class I includes the enzymes from eukaryotes and most archaea, and class II includes the HMG-CoA reductases of certain prokaryotes and archaea. In addition to the divergence in the sequences, the enzymes of the two classes also differ with respect to inhibition by statin drugs. The inhibition constant values for the class I enzymes are nanomolar, whereas the class II reductases are over 4 orders of magnitude less sensitive to inhibition by statins (Table (Table1)1) (1, 12, 27).
The best-studied class I HMG-CoA reductases are those from mammals, plants, yeast, and certain archaea. Eukaryotic HMG-CoA reductases consist of a highly conserved C-terminal catalytic domain and a poorly conserved N-terminal domain that comprises from two to eight transmembrane helices (2). The crystal structure of the catalytic domain of the human enzyme (13) revealed a tetramer with active sites lying at subunit interfaces. The activity of human and other eukaryotic HMG-CoA reductases is regulated by reversible phosphorylation (19), but no eubacterial or archaeal HMG-CoA reductase appears to be regulated by phosphorylation in vivo.
The interest in class II HMG-CoA reductases arises from their presence in certain bacterial pathogens and the discovery that a functional mevalonate pathway is essential for survival of these pathogens (26). The differences in structure, regulation, and sensitivity to statin drugs between the two classes of enzymes suggested, furthermore, that it may be possible to design inhibitors for use as antibiotics that target a class II HMG-CoA reductase. These considerations led to characterization of the class II reductases from Staphylococcus aureus and Enterococcus faecalis in order to supplement the information for the previously characterized class II enzymes from P. mevalonii and Archaeoglobus fulgidus. Inspection of gene assignments by The Institute for Genome Research (www.tigr.org) also suggested that the following bacteria and archaea also encode a class II HMG-CoA reductase: B. burgdorferi, Lactococcus lactis, Listeria innocua, Listeria monocytogenes, Oceanobacillus iheyensis, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Thermoplasma acidophilum, and Thermoplasma volcanium. This review summarizes the properties of the class II HMG-CoA reductases that have been characterized.