A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5′ flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruziHMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.

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Effect of glucose or fructose feeding on cholesterol synthesis in diabetic animals

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1985.249.5.g634 ◽

1985 ◽

Vol 249 (5) ◽

pp. G634-G641 ◽

Cited By ~ 1

Author(s):

K. R. Feingold ◽

A. H. Moser

Keyword(s):

Cholesterol Synthesis ◽

Reductase Activity ◽

Active Form ◽

Diabetic Rats ◽

Hepatic Cholesterol ◽

Hmg Coa Reductase ◽

Hepatic Cholesterol Synthesis ◽

Streptozotocin Induced Diabetes ◽

Intestinal Hypertrophy ◽

Coa Reductase

Previous studies have demonstrated that cholesterol synthesis is increased twofold in the small intestines of rats with streptozotocin-induced diabetes. The purpose of the present study was to determine the effect of adding glucose or fructose to standard rat chow on cholesterol synthesis in control and diabetic rats. In control rats a 25% glucose or fructose diet fed for 21 days markedly inhibited hepatic cholesterol synthesis in the liver. In contrast, in diabetic animals only fructose inhibited hepatic cholesterol synthesis. In both control and diabetic animals the addition of these simple sugars to the diet did not markedly alter extrahepatic cholesterol synthesis. The enhancement of small intestinal cholesterol synthesis observed in diabetic animals was present regardless of the dietary manipulations. Further studies demonstrated that the addition of smaller concentrations of fructose (10%) to standard rat chow decreased hepatic cholesterol synthesis in both control and diabetic rats. Similarly the addition of fructose to the diet of control and diabetics for a period as short as 2 days was also sufficient to inhibit hepatic cholesterol synthesis. In both control and diabetic animals, fructose feeding decreased hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity but did not alter the percentage of HMG-CoA reductase in the active form. Finally, the intestinal hypertrophy and stimulation of intestinal cholesterogenesis that are characteristic of streptozotocin-induced diabetes occurred when either glucose or fructose was the sole caloric source.

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Role of reversible phosphorylation in mediating changes in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase during pregnancy and lactation in the rat

Biochemical Journal ◽

10.1042/bj2480993 ◽

1987 ◽

Vol 248 (3) ◽

pp. 993-996 ◽

Cited By ~ 1

Author(s):

R A Easom ◽

V A Zammit

Keyword(s):

Reductase Activity ◽

Post Partum ◽

Active Form ◽

Total Activity ◽

Female Rats ◽

Hmg Coa Reductase ◽

Pregnancy And Lactation ◽

Coa Reductase

1. The expressed and total (completely dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase were measured in microsomal fractions isolated from cold-clamped liver samples from female rats in various stages of the reproductive cycle. 2. There was little change in total HMG-CoA reductase activity during pregnancy and early lactation, but after 2 days post partum there was a marked increase in total activity. 3. The expressed/total activity ratio of HMG-CoA reductase showed a profound decrease during the last 2 days of pregnancy. The fraction of the enzyme in the active form increased progressively during the first 2 days of lactation. 4. The combined effect of these changes was that the expressed activity of HMG-CoA reductase changed in parallel with the known changes in the hepatic rate of cholesterogenesis during pregnancy and lactation in vivo.

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Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat

Biochemical Journal ◽

10.1042/bj2390285 ◽

1986 ◽

Vol 239 (2) ◽

pp. 285-293 ◽

Cited By ~ 3

Author(s):

R A Smith ◽

B Middleton ◽

D W West

Keyword(s):

Reductase Activity ◽

Active Form ◽

Total Activity ◽

Maximum Activity ◽

Covalent Modification ◽

Light Phase ◽

Hmg Coa Reductase ◽

Enzyme Molecules ◽

Coa Reductase ◽

Homogenization Medium

‘Expressed’ and ‘total’ activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. ‘Expressed’ activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. ‘Total’ activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both ‘expressed’ and ‘total’ activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the ‘expressed’ activity that was close to the nadir value. ‘Expressed’ activity was lower than ‘total’ at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the ‘expressed’/‘total’ activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in ‘expressed’ HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low ‘total’ HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation.

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Production of a Human Metabolite of Atorvastatin by Bacterial CYP102A1 Peroxygenase

Applied Sciences ◽

10.3390/app11020603 ◽

2021 ◽

Vol 11 (2) ◽

pp. 603

Author(s):

Thi Huong Ha Nguyen ◽

Soo-Jin Yeom ◽

Chul-Ho Yun

Keyword(s):

Hydrogen Peroxide ◽

Catalytic Activity ◽

High Throughput Screening ◽

Reductase Activity ◽

High Catalytic Activity ◽

Active Metabolites ◽

Monooxygenase Activity ◽

Highly Active ◽

Statin Drug ◽

Coa Reductase

Atorvastatin is a widely used statin drug that prevents cardiovascular disease and treats hyperlipidemia. The major metabolites in humans are 2-OH and 4-OH atorvastatin, which are active metabolites known to show highly inhibiting effects on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Producing the hydroxylated metabolites by biocatalysts using enzymes and whole-cell biotransformation is more desirable than chemical synthesis. It is more eco-friendly and can increase the yield of desired products. In this study, we have found an enzymatic strategy of P450 enzymes for highly efficient synthesis of the 4-OH atorvastatin, which is an expensive commercial product, by using bacterial CYP102A1 peroxygenase activity with hydrogen peroxide without NADPH. We obtained a set of CYP102A1 mutants with high catalytic activity toward atorvastatin using enzyme library generation, high-throughput screening of highly active mutants, and enzymatic characterization of the mutants. In the hydrogen peroxide supported reactions, a mutant, with nine changed amino acid residues compared to a wild-type among tested mutants, showed the highest catalytic activity of atorvastatin 4-hydroxylation (1.8 min−1). This result shows that CYP102A1 can catalyze atorvastatin 4-hydroxylation by peroxide-dependent oxidation with high catalytic activity. The advantages of CYP102A1 peroxygenase activity over NADPH-supported monooxygenase activity are discussed. Taken together, we suggest that the P450 peroxygenase activity can be used to produce drugs’ metabolites for further studies of their efficacy and safety.

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Effects of diabetes on the expressed and total activities of 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver in vivo. Reversal by insulin treatment

Biochemical Journal ◽

10.1042/bj2300747 ◽

1985 ◽

Vol 230 (3) ◽

pp. 747-752 ◽

Cited By ~ 17

Author(s):

R A Easom ◽

V A Zammit

Keyword(s):

Activity Ratio ◽

Reductase Activity ◽

Active Form ◽

Fold Increase ◽

Total Activity ◽

Diabetic Rats ◽

Diurnal Rhythms ◽

Hmg Coa Reductase ◽

Coa Reductase

The expressed and total activities of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase (EC 1.1.1.34) were measured in microsomal fractions prepared from cold-clamped liver samples [Easom & Zammit (1984) Biochem. J. 220, 733-738] from control or insulin-treated diabetic animals. Streptozotocin-induced diabetes resulted in a marked decrease in total activity of HMG-CoA reductase and in the fraction of the enzyme in the active form, but appreciable effects were only observed in the liver of animals in which the blood glucose was above 20 mM. Intravenous infusion of insulin into diabetic rats resulted in a rapid (less than 20 min) and total dephosphorylation of the enzyme in vivo without any change in total activity. Longer-term (4 h) treatment with insulin (injected intraperitoneally) produced a rapid increase in expressed/total HMG-CoA reductase activity ratio to about 90%, followed, after a lag of 2-3 h, by a 5-6-fold increase in total activity. These observations are discussed with respect to the possible role of insulin in generating and maintaining the respective diurnal rhythms in total and in expressed/total HMG-CoA reductase activity ratio observed for normal animals in vivo [Easom & Zammit (1984) Biochem. J. 220, 739-745].

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Reconstitution of bacterial autotransporter assembly using purified components

eLife ◽

10.7554/elife.04234 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 63

Author(s):

Giselle Roman-Hernandez ◽

Janine H Peterson ◽

Harris D Bernstein

Keyword(s):

Active Form ◽

Single Copy ◽

External Energy ◽

Passenger Domain ◽

Highly Active ◽

Bam Complex ◽

Rate Limiting Step ◽

Necessary And Sufficient ◽

Rate Limiting

Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular (‘passenger’) domain and a C-terminal β barrel (‘β’) domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotransporter EspP in vitro. Our results indicate that the membrane integration of the β domain is the rate-limiting step in autotransporter assembly and that passenger domain translocation does not require the input of external energy. Furthermore, experiments using nanodiscs strongly suggest that autotransporter assembly is catalyzed by a single copy of the Bam complex. Finally, we describe a method to purify a highly active form of the Bam complex that should facilitate the elucidation of its function.

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Acute effects of starvation and treatment of rats with anti-insulin serum, glucagon and catecholamines on the state of phosphorylation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in vivo

Biochemical Journal ◽

10.1042/bj2410183 ◽

1987 ◽

Vol 241 (1) ◽

pp. 183-188 ◽

Cited By ~ 16

Author(s):

R A Easom ◽

V A Zammit

Keyword(s):

Intravenous Injection ◽

Activity Ratio ◽

Reductase Activity ◽

Active Form ◽

Fold Increase ◽

Total Activity ◽

Dark Phase ◽

Hmg Coa Reductase ◽

Coa Reductase

The fraction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the dephosphorylated (active) form in rat liver in vivo was measured after various experimental treatments of animals. Intraperitoneal injection of glucose (to raise serum insulin concentrations) into rats 4 h into the light phase (L-4) resulted in a transient (30 min) increase in the expressed (E)/total (T) activity ratio of HMG-CoA reductase without any change in total activity (obtained after complete dephosphorylation of the enzyme). Conversely, intravenous injection of guinea-pig anti-insulin serum into rats 4 h into the dark phase (D-4) significantly depressed the E/T ratio within 20 min. Intravenous injection of glucagon into normal rats at this time point did not affect the degree of phosphorylation of the enzyme, in spite of a 10-fold increase in hepatic cyclic AMP concentration induced by the hormone treatment. A 3-fold increase in the concentration of the cyclic nucleotide induced by adrenaline infusion was similarly ineffective in inducing any change in expressed or total activities of hepatic HMG-CoA reductase. However, when insulin secretion was inhibited, either by the induction of streptozotocin-diabetes or by simultaneous infusion of somatostatin, glucagon treatment was able to depress the expressed activity of HMG-CoA reductase (i.e. it increased the phosphorylation of the enzyme). Therefore insulin appears to have a dominant role in the regulation of the phosphorylation state of hepatic HMG-CoA reductase. In apparent corroboration of this suggestion, short-term 4 h food deprivation of animals before D-4 resulted in a marked decrease in the E/T activity ratio of reductase, which was not affected further by an additional 8 h starvation. By contrast, the total activity of the enzyme was not significantly affected by 4 h starvation, but was markedly diminished after 12 or 24 h starvation. Longer-term starvation also produced a chronic increase in the degree of phosphorylation of the enzyme. These results are discussed in relation to the role of reversible phosphorylation in the control of hepatic HMG-CoA reductase activity in vivo.

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Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse uterine epithelial cells

Biochemical Journal ◽

10.1042/bj2410279 ◽

1987 ◽

Vol 241 (1) ◽

pp. 279-284 ◽

Cited By ~ 1

Author(s):

L Leijten ◽

P A Wilce ◽

M Davidson ◽

M Banks ◽

L Martin

Keyword(s):

Enzyme Activity ◽

Epithelial Cells ◽

Reductase Activity ◽

Active Form ◽

Cholesterol Feeding ◽

Uterine Epithelial Cells ◽

Coa Reductase ◽

Inhibited Enzyme

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase was studied in mouse uterine epithelium. The enzyme was rapidly inactivated during incubation with ATP/Mg2+ in vitro, and could be re-activated by incubation with partially purified rat liver phosphoprotein phosphatase. Enzyme activity was rapidly inhibited by mevalonate injection in vivo to approx. 30% of control. The percentage of total enzyme active in vivo was measured by inclusion of NaF in the isolation buffers. The percentage of enzyme active in vivo 18 h after stimulation by oestrogens remained at approx. 25% after inhibition of activity by mevalonate injection, cholesterol feeding or progesterone pretreatment. However, 9 h after oestrogen stimulation, cholesterol feeding inhibited enzyme activity to 57% of control, 94% of which was in the active form. We conclude that, although all components for a reversible phosphorylative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity are present in uterine epithelial cells, a role in the rapid changes in epithelial enzyme activity has not been demonstrated.

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Description of a novel eukaryotic deoxyuridine 5′-triphosphate nucleotidohydrolase in Leishmania major

Biochemical Journal ◽

10.1042/bj3250441 ◽

1997 ◽

Vol 325 (2) ◽

pp. 441-447 ◽

Cited By ~ 24

Author(s):

Ana CAMACHO ◽

Rosalía ARREBOLA ◽

Javier PEÑA-DIAZ ◽

Luis M. RUIZ-PÉREZ ◽

Dolores GONZÁLEZ-PACANOWSKA

Keyword(s):

Amino Acid ◽

Rational Design ◽

Leishmania Major ◽

Active Form ◽

Single Copy ◽

Cdna Expression Library ◽

Expression Library ◽

Calculated Molecular Mass ◽

Reading Frame ◽

Highly Active

A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichiacoli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichiacoli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.

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