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PubMed1987

The mechanism of inhibition of Alcaligenes faecalis S-adenosylhomocysteine hydrolase by neplanocin A.

Neplanocin A, a cyclopentenyl analog of adenosine, has been reported by S. Yaginuma, N. Muto, M. Tsujino, Y. Sudate, M. Hayashi, and M. Otari (1981) J. Antibiot. 34, 359-366 to exhibit antibacterial activity against Alcaligenes faecalis. Since neplanocin A (NpcA) is a known inhibitor of eukaryotic S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) (R. T. Borchardt, B. T. Keller, and U. Patel-Thombre (1984) J. Biol. Chem. 259, 4353-4358), the present study was undertaken to determine the effects of this carbocyclic nucleoside on AdoHcy hydrolase isolated from a prokaryotic source (A. faecalis). AdoHcy hydrolase was purified to homogeneity by affinity chromatography on an AdoHcy-agarose matrix from A. faecalis. Neplanocin A inactivated the purified AdoHcy hydrolase in a time- and concentration-dependent manner and the enzyme activity could not be recovered by dialysis. The inactivation of this bacterial enzyme by neplanocin A is accompanied by a reduction of three of the six enzyme-bound NAD+s to NADHs. These results suggest that the prokaryotic enzyme, like the eukaryotic AdoHcy hydrolase, is susceptible to inhibition by neplanocin A. The mechanism of inactivation in both cases appears to be a Kcat mechanism involving the reduction of the enzyme-bound NAD+ to NADH. The fact that total inhibition of the prokaryotic AdoHcy hydrolase by NpcA results in a reduction of only three of the six enzyme-bound NAD+s to NADHs suggests that the enzyme shows half-site reactivity (i.e., only three of the six subunits are catalytically active).

Adenosine↗
PubMed1991

Retinyl ester hydrolases in retinal pigment epithelium.

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.

Animals↗
PubMed1981

beta-hydroxy-beta-methylglutaryl coenzyme A hydrolase of rat liver.

Beta-Hydroxy-beta-methylglutaryl coenzyme A hydrolase, or deacylase, (EC 3.1.2.5) is important, at least potentially, in the regulation of mammalian cholesterol synthesis. This is so for two reasons, both related to the enzyme generally regarded as rate-limiting for cholesterogenesis, namely beta-hydroxy-beta-methylglutaryl CoA reductase: (i) the hydrolase competes for the same substrate as the reductase and (ii) its end product, hydroxymethylglutamic acid, is a known inhibitor of the reductase. Consequently we have examined some of the properties of the hydrolase, as found in rat liver, after first developing a simple isotopic technique for its assay. Beta-Hydroxy-beta-methylglutaryl CoA hydrolase is both soluble and microsomal. The microsomal enzyme is inactivated by pre-incubation at 37 degree C, but not a 4 degree C, has an apparent pH optimum of approximately 7.6, and has Km and V values of 270 (microM) and 33.3 (nmol HMG/10 per mg protein), respectively, at 37 degree C. For the cytosolic enzyme the corresponding Km and V values are 830 and 111.1. From our observations it seems unlikely that beta-hydroxy-beta-methylglutaryl CoA hydrolase plays a significant role in the regulation of hepatic cholesterol synthesis since, in contrast to microsomal beta-hydroxy-beta-methylglutaryl coenzyme A reductase, we could find for the microsomal hydrolase no evidence of a diurnal rhythm of activity, no inhibition of activity by short-term cholesterol feeding and no evidence from Arrhenius-plot data for any membrane-mediated control of enzyme activity. Thus, the significance of the enzyme in mammalian systems remains unknown.

Acyl Coenzyme A↗
PubMed1982

Characterization of neutral and acid ester hydrolase in Wolman's disease.

Wolman's disease is characterized by diffuse cellular accumulation of cholesteryl ester and triacylglycerol, steatorrhea, and death in infancy. Although lysosomal acid ester hydrolase has been reported to be absent in tissues from affected infants, full evaluation of the intracellular ester hydrolases in hepatic and nonhepatic tissues has not been performed previously. Studies on ester hydrolase activity in human liver and skin fibroblasts have permitted the following conclusions. (1) The ester hydrolase activity for cholesteryl oleate and triolein are parallel in human liver and skin fibroblasts. (2) There is a significant loss of activity for both of these substrates at pH 4 in both liver and skin fibroblasts in a subject with Wolman's disease. (3) At pH 7, however, ester hydrolase activity for both substrates in both liver and skin fibroblast preparations from a patient with Wolman's disease is preserved. (4) The patient's mother, an obligate heterozygote, does not demonstrate any loss of activity for either substrate at pH 4. THese data are consistent with the concept that acid and neutral ester hydrolases are different enzymes.

Adult↗
PubMed1984

Inhibition of rat liver retinyl palmitate hydrolase activity by ether analogs of cholesteryl esters and acylglycerides.

In previous studies, retinyl palmitate hydrolase activity in rat liver was partly characterized and was found to correlate and to partially copurify with hydrolytic activities against cholesteryl oleate and triolein. The present studies were designed to further explore relationships between these three lipid ester hydrolase activities, by use of non-hydrolyzable ether analogs of cholesteryl esters and acylglycerides. Cholesteryl ether analogs were potent inhibitors of all three hydrolase activities with relative potencies for the series of ethers of: linoleyl greater than oleyl = palmitoyl greater than n-butyl = n-propyl greater than ethyl = methyl. Retinyl palmitate hydrolase activity was most strongly inactivated by this series of analogs, with 48-86% of the activity inhibited at cholesteryl ether levels of 1 microM. The acylglyceride ether analogs were much weaker inhibitors of the three hydrolase activities, with the triolein, diolein and dipalmitin analogs showing similar inhibitory potencies, greater than that of the monolein and monopalmitin analogs. The data demonstrate the potential usefulness of ether analogs of cholesteryl esters and acylglycerides for exploring some of the characteristics of lipid ester hydrolase activities.

Animals↗
PubMed1985

The regulation of cytosolic epoxide hydrolase in mice.

The concentration of cytosolic epoxide hydrolase in untreated and clofibrate-treated mouse liver extracts was estimated by immunoblotting. Clofibrate treatment of mice was found to increase liver cytosolic epoxide hydrolase concentration by two fold, showing that the increase in cytosolic epoxide hydrolase in mouse liver after clofibrate treatment is primarily due to induction. The induced and uninduced cytosolic epoxide hydrolase, and epoxide hydrolase in the cytosolic and mitochondrial fractions were compared and found to be identical or very similar. Cytosolic epoxide hydrolases in kidney and liver were similar in molecular weight and antigenic properties.

Animals↗
PubMed1989

Substrate specificity of bleomycin hydrolase.

Bleomycin (BLM) hydrolase is believed to protect both malignant and normal tissue from the toxicity of the antitumor drug BLM. Little is known about the substrate specificity of BLM hydrolase. Thus, we developed ion-paired reverse phase high speed liquid chromatography systems to assay for the metabolism of several BLM analogs. We found that BLM A2, BLM B2, tallysomycin S10b (TLM S10b), peplomycin (PEP), butylamino-3-propylamino-3-propylamine bleomycin (BAPP), deglyco bleomycin A2 (dgBLM A2) and bleomycinic acid were each metabolized by rabbit lung BLM hydrolase to a single metabolite. When compared to their corresponding parent compounds, these metabolites were 6- to 35-fold less potent in their ability to inhibit the proliferation of A-253 human head and neck squamous carcinoma cells in culture. Furthermore, we found that substitutions in various regions of the BLM molecule greatly affected the kinetic parameters of BLM hydrolase. For example, the Km with BLM B2 (0.056 +/- 0.005 mM) was 15-fold lower than that seen with BLM A2 (0.83 +/- 0.11 mM). In contrast, the Vmax was not affected markedly by these terminal amine substitutions but was influenced greatly by deletion of the carbohydrate groups of BLM. For example, a 4-fold higher Vmax was observed with dgBLM A2 compared to BLM A2. Thus, these results demonstrate that BLM hydrolase can recognize and metabolize a broad spectrum of BLM analogs regardless of their structural features. This enzymatic conversion resulted in the inactivation of the BLMs as demonstrated by a substantial decrease in their cytotoxicity. Furthermore, the terminal amine and carbohydrate regions, respectively, dictate the apparent affinity and the rate of metabolism of BLM hydrolase substrates.

Animals↗
PubMed1989

Homocysteine potentiates the antiviral and cytostatic activity of those nucleoside analogues that are targeted at S-adenosylhomocysteine hydrolase.

Various adenosine analogues, i.e. (S)-9-(2,3-dihydroxypropyl)adenine, (RS)-3-adenin-9-yl-2-hydroxypropanoic acid, carbocyclic 3-deazaadenosine and neplanocin A, which have been previously recognized as specific inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase, gained a marked increase in their cytostatic activity (against tumor cells) and antiviral activity (against vaccinia and vesicular stomatitis virus) in the presence of L-homocysteine (10(-3) M). Homocysteine did not increase the cytostatic or antiviral activity of those compounds (i.e. tubercidin, ribavirin, acyclovir or vidarabine) that do not achieve their biological activity via SAH hydrolase inhibition. The increased antiviral activity following addition of homocysteine was observed only with those viruses (i.e. vaccinia and vesicular stomatitis virus) that belong to the activity spectrum of SAH hydrolase inhibitors [Biochem Pharmacol 36: 2567-2575, 1987], and only in those cells in which the SAH hydrolase inhibitors are normally active. The enhancing effect of homocysteine on the cytostatic and antiviral activity of the SAH hydrolase inhibitors could not be attributed to a non-specific increase in the cytotoxicity of the compounds, as their effects on host cell macromolecule (DNA, RNA, protein) synthesis was not markedly altered in the presence of homocysteine. Most likely, homocysteine exerted its potentiating effect on the activity of the SAH hydrolase inhibitors through an increase in the intracellular levels of SAH, which is known to act as a product inhibitor of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions.

Adenosine↗
PubMed1991

Influence of the level of cytosolic epoxide hydrolase on the induction of sister chromatid exchanges by trans-beta-ethylstyrene 7,8-oxide in human lymphocytes.

trans-beta-Ethylstyrene 7,8-oxide, a substrate of cytosolic epoxide hydrolase, and 4-fluorochalcone oxide, an inhibitor of this enzyme, were investigated on induction of sister chromatid exchanges (SCE) in human lymphocytes. Both epoxides enhanced the frequency of SCE. 4-Fluorochalcone oxide at low concentration (2.5 microM) inhibited cytosolic epoxide hydrolase activity towards trans-beta-ethylstyrene 7,8-oxide in lymphocytes by 74% and had no effect on glutathione transferase activity using this substrate. At this concentration it did not induce SCE itself, but it potentiated the effect of trans-beta-ethylstyrene 7,8-oxide several fold. In lymphocytes from different subjects, the number of SCE induced by a low concentration of trans-beta-ethylstyrene 7,8-oxide correlated negatively with the individual cytosolic epoxide hydrolase activity (r = -0.72; -0.73 in two series of experiments). The number of SCE induced by a high concentration of trans-beta-ethylstyrene 7,8-oxide did not correlate with cytosolic epoxide hydrolase activity (r = 0.004; -0.24), but a negative correlation was found with glutathione transferase activity (r = -0.50). This finding is consistent with the results of biochemical studies in lymphocytes in which we determined the relative contribution of cytosolic epoxide hydrolase and glutathione transferase to the metabolism of trans-beta-ethylstyrene 7,8-oxide at varying substrate concentrations. The study demonstrates that the level of genotoxic effects induced in human lymphocytes is influenced by the individual level of detoxifying enzymes. At low concentrations, cytosolic epoxide hydrolase was more important than glutathione transferase activity.

Cells, Cultured↗
PubMed1987

Hepatic levels of cytosolic, microsomal and 'mitochondrial' epoxide hydrolases and other drug-metabolizing enzymes after treatment of mice with various xenobiotics and endogenous compounds.

This study was performed in order to study the response of epoxide hydrolases in different subcellular compartments of mouse liver to treatment with various compounds. Male C57BL/6 mice were treated with 31 different compounds--including traditional inducers of xenobiotic-metabolizing systems, liver carcinogens, stilbene derivatives, endogenous compounds and various other drugs and xenobiotics. The effects on liver somatic index; protein contents in 'mitochondria', microsomes and cytosol prepared from the liver; epoxide hydrolase activity towards trans- or cis-stilbene oxide in these three fractions; microsomal cytochrome P-450 content; cytosolic and 'mitochondrial' glutathione transferase activity and cytosolic DT-diaphorase activity were then determined. Cytosolic epoxide hydrolase activity was induced by chlorinated paraffins, di(2-ethylhexyl)phthalate and clofibrate and depressed by alpha-naphthylisothiocyanate, 3-methylcholanthrene, benzil and quercitin. Radial immunodiffusion revealed similar changes in the amount of enzyme protein present, except for two cases, where the increase in amount was larger; and the enzyme seems to be inhibited by benzil. Microsomal epoxide hydrolase activity was induced by these same compounds and several others as well, including dibenzoylmethane, butylated hydroxyanisole and polychlorinated biphenyls. 'Mitochondrial' epoxide hydrolase activity towards trans-stilbene oxide was not affected by those compounds which induced the cytosolic enzyme, but increased about two-fold after treatment with 2-acetylaminofluorene, DL-ethionine, aflatoxin B1 and phenobarbital. There does not seem to be any co-regulation of different forms of epoxide hydrolase in mouse liver. In general small effects were observed on liver weight and protein contents in the different subcellular fractions. Polychlorinated biphenyls were the most potent of the 8 compounds which induced cytochrome P-450, while butylated hydroxyanisole induced cytosolic glutathione transferase activity to the highest extent. 'Mitochondrial' glutathione transferase activity was most induced by certain of the stilbene derivatives. The most potent inducers of DT-diaphorase activity were 3-methylcholanthrene, polychlorinated biphenyls and dinitrotoluene.

Animals↗
PubMed1977

Influence of dietary status and diabetes on aortic acyl-CoA hydrolase activity.

We have postulated that the accelerated snythesis of cholesteryl ester in atherosclerotic microsomes may result in part from decreased acyl-CoA hydrolase activity in arterial tissue, because acyl-CoA is a common substrate for both reactions. We have now investigated the influence of nutritional status, type of diet, and diabetes on the acyl-CoA hydrolase activity of otherwise normal aortic microsomes. Fasting rabbits for 16 hr diminished the acyl-CoA hydrolase activity approximately 30%. The activity of this aortic microsomal enzyme in rats maintained on a high-carbohydrate diet for 5 weeks was comparable to the activity observed on a high fat (olive oil) diet. The type of fat in the diet influences the acyl-CoA hydrolase activity: oils containing 77% oleic acid (high-oleic safflower oil) and containing 70% linoleic acid (conventional safflower oil) lowered the aortic microsomal acyl-CoA hydrolase activity in comparison to a more saturated fat (cocoa butter). Aortic preparations of rats made diabetic by streptozotocin exhibited higher acyl-CoA hydrolase activity than the normal. The results show that conditions associated with human atherogenesis (diabetes and saturated fat diet) increase rather than suppress the activity of this arterial enzyme in normal arterial tissues of the rat.

Animals↗
PubMed1983

Selection and characterization of a murine lymphoid cell line partially deficient in S-adenosylhomocysteine hydrolase.

The exact role of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) in mediating the toxic effects of adenosine toward mammalian cells has not been ascertained. The selection and characterization of S-adenosylhomocysteine hydrolase-deficient cell lines offers a biochemical genetic approach to this problem. In the present experiments, a mutant clone (Sahn 12) with 11-13% of wild-type S-adenosylhomocysteine hydrolase activity was selected from the murine T lymphoma cell line R 1.1 after mutagenesis and culture in adenosine, deoxycoformycin, uridine and homocysteine thiolactone-supplemented medium. In the presence of 0.5 mM homocysteine thiolactone and 10-200 microM adenosine, wild-type and mutant cells synthesized S-adenosylhomocysteine intracellularly at markedly different rates, and excreted the compound extracellularly. Thus, at time points up to 10 h, the S-adenosylhomocysteine hydrolase-deficient lymphoblasts required 5-10-fold higher concentrations of adenosine in the medium to achieve the same intracellular S-adenosylhomocysteine levels as wild-type cells. Similarly, the Sahn 12 lymphoblasts were 5-10-fold more resistant than R 1.1 cells to the toxic effects of adenosine plus homocysteine thiolactone. These results establish that (i) 11-13% of wild-type S-adenosylhomocysteine hydrolase activity is compatible with normal growth, (ii) in medium supplemented with both adenosine and homocysteine thiolactone, intracellular S-adenosylhomocysteine is synthesized by S-adenosylhomocysteine hydrolase, (iii) the net intracellular level of S-adenosylhomocysteine is determined by both the rate of S-adenosylhomocysteine synthesis and its rate of excretion, (iv) under such conditions the accumulation of S-adenosylhomocysteine is related to cytotoxicity, (v) in the absence of an exogenous homocysteine source, S-adenosylhomocysteine derives from endogenous sources, and the accumulation of S-adenosylhomocysteine is not the primary cause of adenosine induced cytotoxicity.

Adenosine↗
PubMed1990

Inhibition of catalase and epoxide hydrolase by the renal cystogen 2-amino-4,5-diphenylthiazole and its metabolites.

Subchronic feeding of 2-amino-4,5-diphenylthiazole (DPT) to rats results in the development of renal cysts and has been used as a model system to study polycystic kidney disease. Because previous studies revealed changes in renal enzymes following DPT administration, a possible direct effect of DPT and its phenolic metabolites on catalase and a related enzyme, epoxide hydrolase, was examined. Experiments with three in vitro systems (suspensions of rabbit renal tubules, rat kidney homogenates, and commercially obtained bovine liver catalase) revealed direct inhibition of catalase activity by the diphenolic metabolite (diOH- DPT: 2-amino-4,5di(4'-hydroxyphenyl)-thiazole), the known renal cystogen nordihydroquaiaretic acid (NDGA) 2-amino-4(4'-hydroxyphenyl),5-phenyl-thiazole (4OH-DPT), and the known catalase inhibitor 3-amino-1,2,4-triazole; DPT did not inhibit catalase activity. Following oral administration to rats of the DPT congeners, 4OH-DPT caused the greatest decrease in both renal catalase and cytosolic epoxide hydrolase activities and the shortest time to onset of cystic lesions. In vitro, mouse liver cytosolic epoxide hydrolase activity was substantially inhibited by 4OH-DPT and dioH-DPT, and NDGA, but not by 2-amino-4-phenyl,5-(4'-hydroxyphenyl)-thiazole (5OH-DPT) or DPT itself. Microsomal epoxide hydrolase (mEH) activity was inhibited by 4OH-DPT, unaffected by DPT or dioH-DPT, and stimulated 2-fold by 5OH-DPT. Finally, mEH activity was substantially higher in samples of normal human kidney than in samples of kidney derived from a patient with autosomal recessive polycystic kidney disease; no differences were observed in cEH activity in these samples. Although the role of altered catalase and epoxide hydrolase activities in cystogenesis is unknown, DPT-induced cyst formation is associated with loss of these enzyme activities in kidney tissue. To our knowledge, this is the first report of an in vivo diminution of cytosolic epoxide hydrolase activity by xenobiotics.

8,11,14-Eicosatrienoic Acid↗
PubMed1981

The effects of modulation of microsomal epoxide hydrolase activity on microsome-catalyzed activation of benzo[alpha]pyrene and its covalent binding to DNA.

The effects of modulation of microsomal epoxide hydrolase activity on the binding of calf thymus DNA of benzo[alpha]pyrene metabolically activated by rat liver microsomes were investigated. In systems where microsomal epoxide hydrolase levels were not manipulated, 2 major bound species, one derived from 9-hydroxybenzo[alpha]pyrene and the other derived from benzo[alpha]pyrene 7,8-dihydrodiol, were found in approximately equivalent amounts. When epoxide hydrolase levels were increased, either by addition in vitro of purified enzyme or by induction in vivo by trans-stilbene oxide, the binding of the benzo[alpha]pyrene 7,8-dihydrodiol product was increased, while the binding of the 9-hydroxybenzo[alpha]pyrene product was practically eliminated. When microsomal epoxide hydrolase activity was decreased by selective inhibition with low concentrations of 1,1,1-trichloropropene 2,3-oxide, the binding of the species derived from 9-hydroxybenzo[alpha]pyrene was increased several-fold, while that of the species derived from benzo[alpha]pyrene 7,8-dihydrodiol was greatly decreased. The results indicate that the binding species derived from 9-hydroxybenzo[alpha]pyrene is formed through a metabolic pathway leading to an epoxide which is a substrate of microsomal epoxide hydrolase and that microsomal epoxide hydrolase is important in regulating the pattern of binding of individual microsomally-formed benzo[alpha]pyrene metabolites to DNA.

Animals↗
PubMed1982

Effect of hyperinsulinemia on acid cholesterol ester hydrolase activity in liver, heart and epididymal fat pad preparations from rats and mice.

The effect of insulin on lysosomal acid cholesterol ester hydrolase activity was studied in liver, heart and fat pad preparations from rats and mice. Hyperinsulinemia was induced for a period of 6 days in rats by the subcutaneous administration of exogenous insulin by an osmotic minipump. The effect of more chronic endogenous hyperinsulinemia was studied using genetic strains of diabetic (db/db) mice at 12 weeks of age. Mouse liver and heart preparations were characterized as having an acid pH optimum of 4.5-5 for cholesterol ester hydrolase activity; a smaller but distinct pH optimum could also be observed at pH 7. In contrast, hydrolase activity in mouse fat pad preparations had only one distinct pH optimum of 6.5. Hyperinsulinemia in rats and mice resulted in a significant decrease in acid cholesterol ester hydrolase activity in heart preparations, but had no consistent effect on acid hydrolase activity observed in liver and fat pad preparations. This decrease in lysosomal acid cholesterol ester hydrolase activity in cardiac tissue due to hyperinsulinemia cannot be related to any changes in lipoprotein turnover caused by insulin or diabetes.

Adipose Tissue↗
PubMed1994

Identification and subcellular localization of leukotriene A4-hydrolase activity in human epidermis.

The purpose of this study was to determine whether normal human epidermis could produce leukotriene B4 (LTB4) from leukotriene A4 (LTA4) ex vivo, and to localize this LTA4-hydrolase activity. Epidermis obtained by suction blister technique incubated with human polymorphonuclear cells, resulted in a 54% increase in LTB4 formation when compared to polymorphonuclear cells incubated alone. Furthermore, human epidermis transformed exogenous LTA4 into LTB4, and this reaction obeyed Michaelis-Menten kinetics with an apparent Km of 6 microM. Subcellular fractionation of homogenized epidermis localized the LTA4-hydrolase activity mainly in the 105,000 x g supernatant fraction (cytoplasmic fraction). This activity was inhibited by two inhibitors of LTA4-hydrolase (bestatin and captopril). Western blot analysis of the 105,000 x g fraction of homogenized epidermis and cultured keratinocytes supported the presence of a LTA4-hydrolase. Thus, normal human epidermis possesses LTA4-hydrolase activity which can transform exogenous LTA4 and polymorphonuclear cell-derived LTA4 into LTB4. The identification of LTA4-hydrolase in the cytoplasmic fraction of human epidermis indicates that epidermal cells may play a more active role in the enzymatic process leading to formation of the proinflammatory compound LTB4 than previously expected.

Blotting, Western↗
PubMed2006

Transition-state complex of the purine-specific nucleoside hydrolase of T. vivax: enzyme conformational changes and implications for catalysis.

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax have previously been solved in complex with inhibitors or a substrate. All these structures show the dimeric T. vivax nucleoside hydrolase with an "open" active site with a highly flexible loop (loop 2) in its vicinity. Here, we present the crystal structures of the T. vivax nucleoside hydrolase with both soaked (TvNH-ImmH(soak)) and co-crystallised (TvNH-ImmH(co)) transition-state inhibitor immucillin H (ImmH or (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol) to 2.1 A and 2.2 A resolution, respectively. In the co-crystallised structure, loop 2 is ordered and folds over the active site, establishing previously unobserved enzyme-inhibitor interactions. As such this structure presents the first complete picture of a purine-specific NH active site, including leaving group interactions. In the closed active site, a water channel of highly ordered water molecules leads out from the N7 of the nucleoside toward bulk solvent, while Trp260 approaches the nucleobase in a tight parallel stacking interaction. Together with mutagenesis results, this structure rules out a mechanism of leaving group activation by general acid catalysis, as proposed for base-aspecific nucleoside hydrolases. Instead, the structure is consistent with the previously proposed mechanism of leaving group protonation in the T. vivax nucleoside hydrolase where aromatic stacking with Trp260 and an intramolecular O5'-H8C hydrogen bond increase the pKa of the N7 sufficiently to allow protonation by solvent. A mechanism that couples loop closure to the positioning of active site residues is proposed based on a comparison of the soaked structure with the co-crystallized structure. Interestingly, the dimer interface area increases by 40% upon closure of loop 2, with loop 1 of one subunit interacting with loop 2 of the other subunit, suggesting a relationship between the dimeric form of the enzyme and its catalytic activity.

Animals↗
PubMed2005

Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev.

Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (+/-)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (+/-)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.

Cryptococcus↗