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.     

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.     

X-ray crystal structures of HMG-CoA synthase from Enterococcus faecalis and a complex with its second substrate/inhibitor acetoacetyl-CoA

Biosynthesis of the isoprenoid precursor, isopentenyl diphosphate, is a critical function in all independently living organisms. There are two major pathways for this synthesis, the non-mevalonate pathway found in most eubacteria and the mevalonate pathway found in animal cells and a number of pathogenic bacteria. An early step in this pathway is the condensation of acetyl-CoA and acetoacetyl-CoA into HMG-CoA, catalyzed by the enzyme HMG-CoA synthase. To explore the possibility of a small molecule inhibitor of the enzyme functioning as a non-cell wall antibiotic, the structure of HMG-CoA synthase from Enterococcus faecalis (MVAS) was determined by selenomethionine MAD phasing to 2.4 A and the enzyme complexed with its second substrate, acetoacetyl-CoA, to 1.9 A. These structures show that HMG-CoA synthase from Enterococcus is a member of the family of thiolase fold enzymes and, while similar to the recently published HMG-CoA synthase structures from Staphylococcus aureus, exhibit significant differences in the structure of the C-terminal domain. The acetoacetyl-CoA binary structure demonstrates reduced coenzyme A and acetoacetate covalently bound to the active site cysteine through a thioester bond. This is consistent with the kinetics of the reaction that have shown acetoacetyl-CoA to be a potent inhibitor of the overall reaction, and provides a starting point in the search for a small molecule inhibitor.     

Multienzyme mevalonate pathway bioreactor

The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.     

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.     

Staphylococcus aureus 3-hydroxy-3-methylglutaryl-CoA synthase: crystal structure and mechanism

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a member of the family of acyl-condensing enzymes, catalyzes the first committed step in the mevalonate pathway and is a potential target for novel antibiotics and cholesterol-lowering agents. The Staphylococcus aureus mvaS gene product (43.2 kDa) was overexpressed in Escherichia coli, purified to homogeneity, and shown biochemically to be an HMG-CoA synthase. The crystal structure of the full-length enzyme was determined at 2.0-A resolution, representing the first structure of an HMG-CoA synthase from any organism. HMG-CoA synthase forms a homodimer. The monomer, however, contains an important core structure of two similar alpha/beta motifs, a fold that is completely conserved among acyl-condensing enzymes. This common fold provides a scaffold for a catalytic triad made up of Cys, His, and Asn required by these enzymes. In addition, a crystal structure of HMG-CoA synthase with acetoacetyl-CoA was determined at 2.5-A resolution. Together, these structures provide the structural basis for an understanding of the mechanism of HMG-CoA synthase.     

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.     

Regulation of early enzymes of ergosterol biosynthesis in Saccharomyces cerevisiae.

In order to determine the regulation mechanisms of ergosterol biosynthesis in yeast, we developed growth conditions leading to high or limiting ergosterol levels in wild type and sterol-auxotrophic mutant strains. An excess of sterol is obtained in anaerobic sterol-supplemented cultures of mutant and wild type strains. A low sterol level is obtained in aerobic growth conditions in mutant strains cultured with optimal sterol supplementation and in wild type strain deprived of pantothenic acid, as well as in anaerobic cultures without sterol supplementation. Measurements of the specific activities of acetoacetyl-CoA thiolase, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase (the first three enzymes of the pathway), show that in cells deprived of ergosterol, acetoacetyl-CoA thiolase and HMG-CoA synthase are generally increased. In an excess of ergosterol, in anaerobiosis, the same enzymes are strongly decreased. A 5-10-fold decrease is observed for acetoacetyl-CoA thiolase and HMG-CoA synthase. In contrast, HMG-CoA reductase is only slightly affected by these conditions. These results show that ergosterol could regulate its own synthesis, at least partially, by repression of the first two enzymes of the pathway. Our results also show that exogenous sterols, even if strongly incorporated by auxotrophic mutant cells, cannot suppress enzyme activities in aerobic growth conditions. Measurement of specific enzyme activities in mutant cells also revealed that farnesyl pyrophosphate thwarts the enhancement of the activities of the two first enzymes.     

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631)

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.     

Effect of phenylmethylsulfonyl fluoride on sterol biosynthesis in 10,000 x g supernatant fraction of rat liver homogenates

Phenylmethylsulfonyl fluoride (PMSF), a reagent commonly employed for the inhibition of serine proteases, has been found to cause significant inhibition of the incorporation of labeled acetate, but not mevalonate, into nonsaponifiable lipid and digitonin-precipitable sterols in the 10,000 X g supernatant fraction of rat liver homogenate preparations. In two experiments, the extent of inhibition of the synthesis of digitonin-precipitable sterols from acetate by PMSF at 1 mM was 81 and 65%. PMSF inhibited the synthesis of nonsaponifiable lipid from acetate at concentrations as low as 0.1 microM. Preincubation of the 10,000 X g supernatant fraction of rat liver homogenates with PMSF (1 mM) resulted in a significant reduction of the activities of acetate thiokinase and 3-hydroxy-3-methylglutaric acid (HMG)-CoA synthase, but did not affect the activities of acetoacetyl-CoA thiolase. Preincubation of rat liver microsomes with PMSF (1 mM) caused a 50% reduction in the level of HMG-CoA reductase activity. The combined results indicate that major sites of action of PMSF in the inhibition of sterol biosynthesis from labeled acetate appear to be on the activities of acetate thiokinase, HMG-CoA synthase, and HMG-CoA reductase. Another reagent used to inhibit serine proteases, diisopropylfluorophosphate, had (at a concentration of 1 mM) no effect on the activities of cytosolic acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase.     

Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/-) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/- embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/- mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/- and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.     

Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase in a tobacco mutant that overproduces sterols.

In a tobacco mutant callus, containing up to tenfold more sterols than the wild-type genotype, HMG-CoA reductase activity is increased by a factor of approximately three, as is the case in mutant seedlings and plants. The rate of HMG-CoA synthesis from acetyl-CoA by the coupled enzyme system acetoacetyl-CoA thiolase/HMG-CoA synthase, as well as its conversion to acetyl-CoA plus acetoacetate by action of HMG-CoA lyase are not affected. These results confirm the key-regulating role of HMG-CoA reductase in sterol biosynthesis, which seems not to be confined only to the animal kingdom, but can also be extended to plants.     

Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.     

Inhibitors of sterol synthesis. Effects of dietary 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on early enzymes in hepatic cholesterol biosynthesis

The effects of dietary administration (0.1% in diet for 8 days) of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on the levels of activity of cytosolic acetoacetyl coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, and microsomal HMG-CoA reductase in liver have been studied in male Sprague-Dawley rats. Significant increases in the levels of activity of acetoacetyl-CoA thiolase and of HMG-CoA synthase were observed. The levels of microsomal HMG-CoA reductase activity were increased, relative to pair-fed control animals, in three experiments and increased, relative to ad libitum control animals, in one of three experiments. When compared with other agents for which the primary mode of action is an inhibition of the intestinal absorption of cholesterol, the magnitude of the increases in the levels of hepatic microsomal HMG-CoA reductase activity in the 15-ketosterol-fed rats was considerably smaller. In view of the previously described marked activity of the 15-ketosterol in the inhibition of the intestinal absorption of cholesterol, as well as its known effects in lowering HMG-CoA reductase activity in mammalian cells in culture, it is proposed that the 15-ketosterol may suppress the elevated levels of hepatic microsomal HMG-CoA reductase activity induced by the reduced delivery of cholesterol to liver as a consequence of the inhibition of the intestinal absorption of cholesterol.     

The structure of the catalytic portion of human HMG-CoA reductase

In higher plants, fungi, and animals isoprenoids are derived from the mevalonate pathway. The carboxylic acid mevalonate is formed from acetyl-CoA and acetoacetyl-CoA via the intermediate 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). The four-electron reduction of HMG-CoA to mevalonate, which utilizes two molecules of NADPH, is the committed step in the biosynthesis of isoprenoids. This reaction is catalyzed by HMG-CoA reductase (HMGR). The activity of HMGR is controlled through synthesis, degradation and phosphorylation. The human enzyme has also been targeted successfully by drugs, known as statins, in the clinical treatment of high serum cholesterol levels. The crystal structure of the catalytic portion of HMGR has been determined recently with bound reaction substrates and products. The structure illustrates how HMG-CoA and NADPH are recognized and suggests a catalytic mechanism. Catalytic portions of human HMGR form tight tetramers, explaining the influence of the enzyme's oligomeric state on the activity and suggesting a mechanism for cholesterol sensing.     

Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia

The effects on cholesterol biosynthesis of growth of cultured C-6 glial cells in serumfree medium ± supplementation with linoleic or linolenic acid were studied. Markedly higher activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) were observed in cells grown in linoleate- or linolenate-supplemented versus nonsupplemented medium. After 48 h HMG-CoA reductase activities were two-and four-fold higher in cells supplemented with 20 and 100 μm linoleate, respectively. The increase in activity became apparent after 24 h and was marked after 48 h. Rates of incorporation of [¹⁴C]acetate or ³H₂O into sterols did not reflect the changes in reductase activity. Thus, in cells supplemented with 50 μm linoleate for 24 and 48 h rates of incorporation of [¹⁴C]acetate were 75–80% lower than rates in nonsupplemented cells. This difference resulted because over the first 24 h of the experiment a fivefold increase in the rate of sterol synthesis occurred in the nonsupplemented cells, whereas essentially no change occurred in the linoleate-supplemented cells; little further change occurred between 24 and 48 h in the nonsupplemented and the linoleate-supplemented cells. That the difference in sterol synthesis under these experimental conditions could be mediated at the level of HMG-CoA synthase (EC 4.1.3.5) was suggested by two series of findings, i.e., first, similar quantitative and temporal changes in the activity of this enzyme, and, second, no change in the activity of acetoacetyl-CoA thiolase (EC 2.3.1.9) or the incorporation of [¹⁴C]mevalonate into sterols. Thus, the data suggest that HMG-CoA synthase, and not HMG-CoA reductase, may direct the rate of cholesterol biosynthesis under these conditions of serum-free growth ± supplementation with polyunsaturated fatty acid.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis by a non-sterol mevalonate-derived product in Mev-1 cells. Apparent translational control

A somatic cell mutant (Mev-1) auxotrophic for mevalonate by virtue of a complete lack of detectable 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activity has been shown to demonstrate a requirement for a non-sterol mevalonate-derived product for regulation of synthesis of HMG-CoA reductase. A comparison of the effects of 25-hydroxycholesterol and the combination of 25-hydroxycholesterol and mevalonate on HMG-CoA reductase activity, synthesis, and mRNA levels in Mev-1 is presented in this report. The results show a close correlation between activity, rate of synthesis, and mRNA levels for Mev-1 cells treated with 25-hydroxycholesterol alone. Under the conditions of these experiments these effects are relatively small (approximately a 4-fold decrease). A much larger inhibition of HMG-CoA reductase activity and rate of synthesis (approximately 50-fold) is observed upon treatment of Mev-1 cells with a combination of 25-hydroxycholesterol and mevalonate. Yet, under these conditions mRNA levels are still reduced by only a factor of 4. These results are interpreted to suggest that the non-sterol mevalonate-derived regulatory product of HMG-CoA reductase acts by a translational control mechanism.     

Mevinolin-resistant mutations identify a promoter and the gene for a eukaryote-like 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the archaebacterium Haloferax volcanii.

Both eukaryotes and archaebacteria use 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase to synthesize mevalonate, which eukaryotes employ in the production of sterols and archaebacteria need for the isoprenoid side chains of their unique and characteristic lipids. The drug mevinolin inhibits HMG-CoA reductase in eukaryotes and in the halophilic archaebacteria, and we have used a spontaneous mutation to mevinolin resistance in the construction of a selectable shuttle vector for Haloferax volcanii. Sequence analysis shows that this resistance determinant encodes an HMG-CoA reductase very like its eukaryotic homologs, but sharing with the one sequenced eubacterial HMG-CoA reductase (that of Pseudomonas mevalonii) few residues other than those common to all HMG-CoA reductases. Characterization of several spontaneous mevinolin-resistant mutants reveals that they are of two sorts: amplifications of the HMG-CoA reductase gene with varying amounts of flanking sequence, and point mutants upstream of the HMG-CoA reductase coding region. We compared sequence and expression of a mutant gene of the latter class to those of the wild-type gene. The point mutation found affects the TATA box-like "distal promoter element," results (like gene amplification) in resistance through the synthesis of excess gene product, and provides the first true genetic definition of an archaebacterial promoter.     

A structural limitation on enzyme activity: the case of HMG-CoA synthase

Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine. Subsequent enzymatic studies showed that the overall reaction rate of the enzyme was increased 140-fold. An X-ray crystallographic study of the Ala110Gly mutant enzyme demonstrated unanticipated adjustments in the active site that resulted in additional stabilization of all three steps of the reaction pathway. The rates of acetylation and hydrolysis of the mutant enzyme increased because the amide nitrogen of Ser308 shifts 0.4 A toward the catalytic cysteine residue. This motion positions the nitrogen to better stabilize the intermediate negative charge that develops on the carbonyl oxygen of the acetyl group during both the formation of the acyl-enzyme intermediate and its hydrolysis. In addition, the hydroxyl of Ser308 rotates 120 degrees to a position where it is able to stabilize the carbanion intermediate formed by the methyl group of the acetyl-S-enzyme during its condensation with acetoacetyl-CoA.