Isolation and characterization of yeast mutants blocked in mevalonic acid formation

Yeast mutants defective in beta-hydroxy-beta-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase have been isolated. Mutants impaired in acetoacetyl-CoA thiolase range into two linked complementation units, erg 10 A and erg 10 B. Mutants deficient in beta-hydroxy-beta-methylglutaryl-CoA synthase belong to two unlinked complementation groups, erg 11 and erg 13. In strictly anaerobic growth conditions, mutants impaired in beta-hydroxy-beta-methylglutaryl-CoA synthase require mevalonic acid in addition to sterol and oleic acid, pointing out the role of mevalonic acid in other physiological function than ergosterol precursor. Growth of mutants impaired in acetoacetyl-CoA thiolase cannot be recovered by mevalonic acid supplementation, suggesting a role of acetoacetyl-CoA or thiolase not linked to sterol pathway.     

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.     

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.     

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.     

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.     

Regulation of hepatic cholesterol biosynthesis by fatty acids: effect of feeding olive oil on cytoplasmic acetoacetyl-coenzyme A thiolase, beta-hydroxy-beta-methylglutaryl-CoA synthase, and acetoacetyl-coenzyme A ligase

We reported previously that, in the perfused rat liver, oleic acid increased the specific activity of cytosolic enzymes of cholesterol biosynthesis. In this study, we examined the effects of oral administration of olive oil on the activities of HMG-CoA synthase, AcAc-CoA thiolase, AcAc-CoA ligase and HMG-CoA reductase. Olive oil feeding increased the specific activity of hepatic HMG-CoA synthase by 50%, AcAc-CoA thiolase by 2-fold, and AcAc-CoA ligase by 3-fold. Olive oil had no effect on HMG-CoA reductase activity. These data suggest that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are regulated in parallel by a physiological substrate, fatty acid, independent of HMG-CoA reductase under these conditions.     

Enzymes involved in the metabolism of 3-hydroxy-3-methylglutaryl-coenzyme A in Catharanthus roseus

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) is an important intermediate in various metabolic pathways, e.g. sterol biosynthesis, ketogenesis and leucine catabolism. The reactions and enzymes involved in the metabolism of HMG-CoA are briefly reviewed. These enzymes have been studied in Catharanthus roseus, a model system for studies on the regulation of secondary metabolic pathways, particularly those leading to terpenoidindole alkaloids. By using HPLC, three HMG-CoA catabolizing enzyme activities have been detected in protein extracts from suspension cultured C. roseus cells: HMG-CoA lyase, 3-nucleotidase and (tentatively identified) 3-methylglutaconyl-CoA hydratase (HMG-CoA hydrolyase). The enzymes have been partially purified. HMG-CoA is formed from three molecules of acetyl-CoA, via reactions which are catalyzed by two (as in yeast and animal cells, via intermediacy of acetoacetyl-CoA) or by just one enzyme (as in e.g. radish). It is yet not clear which process occurs in C. roseus.Abbreviations AACT acetoacetyl-CoA thiolase - AACT/HMGS acetoacetyl-COA thiolase/HMG-CoA synthase - CoASH coenzyme A (reduced form) - HMG-CoA 3-hydroxy-3-methylglutaryl-CoA - MG-CoA 3-methylglutaconyl-CoA     

Location and regulation of early enzymes of sterol biosynthesis in yeast.

Acetoacetyl CoA thiolase and 3-hydroxy-3-methylglutaryl (HMG) CoA synthase were found almost entirely in the cytosol of Saccharomyces cerevisiae, whereas HMG CoA reductase was found almost entirely in mitochondria and further located in the matrix. Formation of all three enzymes was inhibited by cycloheximide, but not by chloramphenicol, indicating that they were synthesized in the cytosol. In anaerobically growing cells the levels of acetoacetyl CoA thiolase and HMG CoA synthase were decreased by ergosterol, whereas HMG CoA reductase levels were affected only slightly, suggesting that in yeast the enzymes responsible for synthesis of HMG CoA were regulated by ergosterol. Aerobically growing cells were essentially impermeable to ergosterol and cholesterol, whereas those growing anaerobically and requiring sterols were readily permeable. Mutants blocked in ergosterol formation were also permeable to sterols under aerobic conditions.     

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.     

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.     

Peroxisomal protein targeting and identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. Recently, it has been demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biogenesis that previously were considered to be cytosolic or located in the endoplasmic reticulum. Peroxisomes have been shown to contain acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase and FPP synthase. Moreover, the activities of these enzymes are also significantly decreased in liver tissue and fibroblast cells obtained from patients with peroxisomal deficiency diseases. In addition, the cholesterol biosynthetic capacity is severely impaired in cultured skin fibroblasts obtained from patients with peroxisomal deficiency diseases. These findings support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis. This paper presents a review of peroxisomal protein targeting and of recent studies demonstrating the localization of cholesterol biosynthetic enzymes in peroxisomes and the identification of peroxisomal targeting signals in these proteins.     

Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase.

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.     

Target of inhibition by the anti-lipogenic antibiotic cerulenin of sterol synthesis in yeast

The antibiotic cerulenin inhibited the incorporation of ¹⁴C-acetyl-CoA by 67% at a concentration of 9 × 10^?6 M but not that of ¹⁴C-HMG-CoA into the non-saponifiable fraction in a cell-free extract of Saccharomyces cerevisiae. Cerulenin markedly inhibited the activity of partially purified HMG-CoA synthase. No inhibition of acetoacetyl-CoA thiolase activity was observed in the same preparation of HMG-CoA synthase. Therefore, cerulenin inhibition of overall sterol synthesis may be accounted for by the specific inhibition of HMG-CoA synthase activity.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

The effect of mevinolin on cytosolic acetoacetyl coenzyme a thiolase activity in Chinese hamster ovary fibroblasts

The effects of mevinolin on cytosolic acetoacetyl CoA thiolase activity were studied in wild type Chinese hamster ovary fibroblasts and in CHO cells adapted to growth in high levels of mevinolin. Acetoacetyl CoA thiolase, HMG CoA synthase and HMG CoA reductase activities were elevated in the mevinolin resistant line, KH 2.0. Thiolase activity was also increased when wild type cells were incubated for 5 days with 1 micron mevinolin. These results are consistent with the hypothesis that the regulation of the first three enzymes in the cholesterol biosynthetic pathway is mediated at least in part via a common mechanism.     

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.     

Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes.

Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type.     

Enzymes involved in acetoacetate formation in various bovine tissues

1. The activities of acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase and lyase and acetoacetyl-CoA deacylase were measured in homogenates of samples of liver, rumen epithelium (long papillae), kidney and lactating mammary gland derived from slaughtered cows. 2. The activities of the four enzymes in bovine liver were similar to the activities previously reported for the corresponding enzymes in rat liver. 3. Acetoacetyl-CoA thiolase and hydroxymethylglutaryl-CoA synthase and lyase were present in rumen epithelium. The activities of the enzymes were all lower on a wet weight basis than in liver. Only very slight deacylase activity was detected. 4. Kidney contained acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA lyase and acetoacetyl-CoA deacylase, but only trace amounts of hydroxymethylglutaryl-CoA synthase. 5. Mammary gland contained acetoacetyl-CoA thiolase and some hydroxymethylglutaryl-CoA lyase, but virtually no hydroxymethylglutaryl-CoA synthase or acetoacetyl-CoA deacylase. 6. Since physiologically significant ketogenesis probably occurs solely via the hydroxymethylglutaryl-CoA pathway, it is evident that, of the four tissues examined, such ketogenesis must be restricted to the liver and the rumen epithelium. 7. All the enzymes except hydroxymethylglutaryl-CoA lyase were also assayed in the four tissues derived from cows suffering from bovine lactational ketosis. Ketosis did not cause a statistically significant change in the activity of any of the enzymes measured. 8. Hepatic hydroxymethylglutaryl-CoA synthase and lyase were found to be associated mainly with the particulate fraction, as in the rat. A considerably greater proportion of these enzymes was found to be present in the cytoplasmic fraction from rumen epithelium, although it was not excluded that this was due to mitochondrial damage during homogenization. 9. Appreciable hydroxymethylglutaryl-CoA synthase was also present in epithelium from the dorsal region of the rumen, from the reticulum and from the omasum, but not from the abomasum.     

Physiological roles of acetoacetyl-CoA thiolase in n-alkane-utilizable yeast, Candida tropicalis: possible contribution to alkane degradation and sterol biosynthesis.

The presence of two types of thiolases, acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase, was demonstrated in peroxisomes of n-alkane-grown Candida tropicalis [Kurihara, T., Ueda, M., & Tanaka, A. (1989) J. Biochem. 106, 474-478], while acetoacetyl-CoA thiolase was also shown to be present in cytosol. The activity of the enzyme in cytosol was constant irrespective of culture conditions, while the peroxisomal enzyme was inducibly synthesized in the alkane-grown yeast cells. These results indicate that peroxisomal acetoacetyl-CoA thiolase participates in alkane degradation, while the cytosolic enzyme is associated with other fundamental metabolic processes, probably sterol biosynthesis, because this enzyme can catalyze the first step of the sterol biosynthesis. 3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase, a key regulatory enzyme of sterol biosynthesis, was found to be localized exclusively in microsomes of the alkane-grown yeast cells. These results suggest that yeast peroxisomes do not contribute to sterol biosynthesis, unlike the case of mammalian cells.     

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.