Showing papers on "Esterase published in 1998"
TL;DR: In vitro evolution is used to probe the relationship between stability and activity in a mesophilic esterase and indicates that these properties are not inversely correlated, which is a positive sign for enhanced thermostability.
Abstract: We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14°C increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.
538 citations
TL;DR: The segregation of HEF's three functions into structurally distinct domains suggests that the entire stem region, including sequences at the amino and carboxy termini ofHEF1 which precede the post-translational cleavage site between HEF1 and HEF2, forms an independent fusion domain which is probably derived from an ancestral membrane fusion protein.
Abstract: The spike glycoproteins of the lipid-enveloped orthomyxoviruses and paramyxoviruses have three functions: to recognize the receptor on the cell surface, to mediate viral fusion with the cell membrane, and to destroy the receptor. In influenza C virus, a single glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, possesses all three functions. In influenza A and B, the first two activities are mediated by haemagglutinin and the third by a second glycoprotein, neuraminidase. Here we report the crystal structure of the HEF envelope glycoprotein of influenza C virus. We have identified the receptor-binding site and the receptor-destroying enzyme (9-O-acetylesterase) sites, by using receptor analogues. The receptor-binding domain is structurally similar to the sialic acid-binding domain of influenza A haemagglutinin, but binds 9-O-acetylsialic acid. The esterase domain has a structure similar to the esterase from Streptomyces scabies and a brain acetylhydrolase. The receptor domain is inserted into a surface loop of the esterase domain and the esterase domain is inserted into a surface loop of the stem. The stem domain is similar to that of influenza A haemagglutinin, except that the triple-stranded, alpha-helical bundle diverges at both of its ends, and the amino terminus of HEF2, the fusion peptide, is partially exposed. The segregation of HEF's three functions into structurally distinct domains suggests that the entire stem region, including sequences at the amino and carboxy termini of HEF1 which precede the post-translational cleavage site between HEF1 and HEF2, forms an independent fusion domain which is probably derived from an ancestral membrane fusion protein.
227 citations
TL;DR: Observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.
Abstract: A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2% N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45 degrees C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity when p-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.
185 citations
TL;DR: The PAD gene (pad) is transcriptionally regulated by p-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD.
Abstract: Phenolic acids, also called substituted cinnamic acids, are important lignin-related aromatic acids and natural constituents of plant cell walls. These acids (particularly ferulic, p-coumaric, and caffeic acids) bind the complex lignin polymer to the hemicellulose and cellulose in plants (1) or are generally esterified with tartaric acid (for example, in grape must, wine, and cider) and can be released as free acids during wine making by some cinnamoyl esterase activities (9). Most often, free phenolic acids are metabolized by different microorganisms into 4-vinyl derivatives and then are eventually reduced into 4-ethyl derivatives (5, 6). Some of these volatile phenols, particularly vinyl and ethyl guaiacol (generated from ferulic acid), are useful aromatic chemicals (12) or contribute naturally to aroma in wine (10) and other fermented foods and beverages. Other volatile phenols, such as ethyl and vinyl phenols (from p-coumaric acid), are most often considered phenolic off-flavors and are responsible for alterations in organoleptic properties. Previously, only three bacterial phenolic acid decarboxylases (PADs) have been purified and characterized (4, 8, 13). Two of these enzymes have been cloned and sequenced, a ferulate decarboxylase (FDC) from Bacillus pumilus (5) and a p-coumarate decarboxylase (PDC) from Lactobacillus plantarum (17). Although these enzymes exhibit 66% amino acid sequence identity, they differ in structure, biochemical characteristics, and substrate specificity. They are also different from the phenylacrylic decarboxylase cloned from Saccharomyces cerevisiae (7), which exhibited very low activity with ferulic and p-coumaric acids. The substrate specificity of these bacterial decarboxylases (ferulic and p-coumaric acids for FDC and p-coumaric and caffeic acids for PDC) is an obstacle for production of aroma compounds from crude or partially purified substrates, which always contain these two acids. It was our goal to screen new microorganisms in order to isolate decarboxylases with different substrate specificities and to better characterize this enzyme family. A comparison of amino acid sequences should help identify regions that specify substrate specificity and residues essential for catalysis. The results presented here are a first step toward obtaining recombinant enzymes with appropriate substrate specificities for aroma production and toward engineering genetically modified starters for vegetable fermentation and wine making. In the course of our screening, we found that Bacillus subtilis was able to decarboxylate ferulic, p-coumaric, and caffeic acids. We describe the cloning and the results of a transcriptional analysis of a pad gene that encodes a PAD. Purification and characterization of the stable recombinant enzyme overexpressed in Escherichia coli confirmed that B. subtilis PAD can metabolize all three phenolic acids; to our knowledge, this is a novel substrate specificity for an enzyme belonging to the PAD family. The PAD examined, which exhibits extensive similarity to FDC in amino acid sequence and differs from FDC in enzymatic characteristics, should be useful in experiments to determine substrate specificity and in catabolic site characterization studies in which site-directed mutagenesis is used.
156 citations
TL;DR: The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated and mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones.
Abstract: The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.
152 citations
TL;DR: The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1) on the basis of substrate specificity and the action of inhibitors, and supported the previous suggestion of a catalytic triad made up of Ser-His-Asp.
Abstract: We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.
142 citations
TL;DR: The amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity, and it is concluded that the balance of these two enzyme activities is important for isoamy acetate accumulation in sake mash.
Abstract: Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash
118 citations
TL;DR: The Tween 80 assay to detect lipolytic activities in agar media was evaluated and the activity was similar to that obtained with p-nitrophenyl butyrate; the enzyme activities were between the esterase and lipase categories.
Abstract: The Tween 80 assay to detect lipolytic activities in agar media was evaluated A spectrophotometric assay for Tween 80 hydrolysis was established The specific activities with Tween 80, as well as with some conventional lipase-type and esterase-type substrates, were measured using several lipases and esterases The activity with Tween 80 was similar to that obtained with p-nitrophenyl butyrate; the enzyme activities with both substrates were between the esterase and lipase categories © Rapid Science Ltd 1998
86 citations
TL;DR: A genomic library of the hyperthermophilic archaeon Pyrococcus furiosus was constructed in Escherichia coli using pBluescript II SK(+) as a cloning vector and tests of esterase activity showed the highest temperature optimum and thermostability among esterases reported to date.
Abstract: A genomic library of the hyperthermophilic archaeon Pyrococcus furiosus was constructed in Escherichia coli using pBluescript II SK(+) as a cloning vector. One positive clone exhibiting thermophilic ester-hydrolyzing activity was directly detected by an in situ plate assay using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-acetate. The plasmid isolated from the clone contained a 3.8 kb HindIII fragment from P. furiosus. Expression of active thermostable esterase in E. coli was independent of isopropyl-β-d-thiogalactopyranoside, suggesting that the archaeal esterase gene was heterologously controlled by its own promoter sequence, not by the vector-located lac promoter. Assays of esterase activity in heat-treated extract of the recombinant E. coli showed the highest temperature optimum (100°C) and thermostability (a half-life of 50 min at 126°C) among esterases reported to date. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 624-629, 1998.
81 citations
TL;DR: A recombinant esterase from Pseudomonas fluorescens was produced from Escherichia coli cultures and purified to homogeneity resulting in a specific activity of 120 U mg −1, which showed high rate and enantioselectivity in the resolution of α-phenyl ethanol and its acetate.
78 citations
TL;DR: Two isolates of Trichoderma harzianum were tested for their capacity to reduce the level of hydrolytic enzymes produced by Botrytis cinerea both in vitro and in vivo, and to inhibit infection caused by B. cine Andrea conidia.
TL;DR: In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme, PME, and showed full identity to the sequenced peptides.
Abstract: Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point >9, a pH optimum at 7 and temperature optimum at 50 °C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with K
m values of 0.7 mg ml-1 for pectin with a DE of 70% and 17 mg ml-1 for pectin with a DE of 25%. The sequences of the NH2-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands.
TL;DR: It can be concluded that the rat may not be a suitable choice for oral bioavailability studies of ester prodrugs; it may be advantageous to target esterProdrugs of hydrophilic compounds to the colon, thus preventing significant accumulation of the parent compound inside the mucosal cells.
TL;DR: Comparison of the amino acid sequence of this enzyme allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coli esterase, suggesting that the enzyme exists in a monomeric form.
Abstract: An esterase from Escherichia coli that is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaC gene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 degreesC and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coli esterase.
TL;DR: An increased activity of a MCE in the R strain is the probable major mechanism conferring resistance to malathion in A. calandrae, the first characterization of a biochemical resistance mechanism in a parasitoid with a high level of resistance to an organophosphate insecticide.
TL;DR: It is proposed that the patatin-like hydrolase is involved in lipid body mobilization as maximal amounts of the protein were found at an early stage of mobilization and confined to lipid bodies.
TL;DR: The feruloyl esterase activities reported to date are predominantly microbial, and these types of enzymes are secreted by several species to facilitate breakdown of the plant cell wall as a food source.
Abstract: Specificity of ferulic acid (feruloyl) esterases G. Williamson’, C. B. Faulds and P. A. Kroon Biochemistry Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. 205 moyl esters (manifest as a K,,, of between lop6 Feruloyl esterases are a subclass of the carboxylic ester hydrolases (EC 3.1.1.1) which hydrolyse ester linkages between hydroxycinnamates and sugars. These types of ester linkage are found in a wide variety of plant cell walls [ 11. The feruloyl esterase activities reported to date are predominantly microbial (Table l ) , and these types of enzymes are secreted by several species to facilitate breakdown of the plant cell wall as a food source. Although there are many types of esterase, the unique feature of the feruloyl esterases is their specificity for hydrolysis of hydroxycinna-
TL;DR: Comamonas acidovorans strain TB-35 has an esterase that degrades solid polyester polyurethane (PUR), which has now been cloned in Escherichia coli and found similarity in the number and positions of cysteine and salt bonds was very apparent between PudA and T AChE.
TL;DR: A model is proposed in which ester synthesis is a consequence of the arrest of lipid biosynthesis resulting from a lack of oxygen and acyl esters are formed as secondary products of reactions aimed at recovering free coenzyme A.
Abstract: During anaerobic fermentation, Saccharomyces cerevisiae releases large amounts of medium-chain fatty acids (MCFAs) and related ethyl esters which are very important for aromatic quality of fermented beverages. The physiological function of ester synthesis is not yet understood. As MCFAs are toxic, their conversion to esters has been proposed to be a detoxification mechanism. Esterases possess ester synthesizing ability. Throughout an anaerobic fermentation of a lipid-free synthetic medium carried out with a S. cerevisiae strain selected for wine making, we have monitored MCFA and ethyl ester production and, at the same time, measured growth and esterasic activity of intact cells. Because no correlation was found between the concentration of each fatty acid and its ethyl ester, there is no evidence that ester synthesis reduces the toxicity of MCFAs. Esterasic activity did not show any correlation with ester synthesis, but it was related to the release of MCFAs. A model is proposed in which ester synthesis is a consequence of the arrest of lipid biosynthesis resulting from a lack of oxygen. Under these conditions, an excess of acyl coenzyme A is produced, and acyl esters are formed as secondary products of reactions aimed at recovering free coenzyme A.
TL;DR: The presence of neuropathy target esterase in essentially all neurons indicates that the selective vulnerability of long axons to neuropathic organophosphates is dependent on factors additional to the presence of the target protein.
TL;DR: The data show that human and rat liver fractions were more active than human duodenal mucosa and human blood leukocytes at hydrolysing the compounds, and the rank order of the compounds was, however, very similar in the different biological systems.
Abstract: One way to minimise systemic side effects of drugs is to design molecules, soft drugs, in such a way that they are metabolically inactivated rapidly after having acted on their pharmacological target. Hydrolases (esterases, peptidases, lipases, glycosidases, etc.) are enzymes well suited to use for drug inactivation since they are ubiquitously distributed. Insertion of ester bonds susceptible to enzymatic cleavage may represent one approach to make the action of a drug more restricted to the site of application. The present study describes the chemical synthesis of fourteen model compounds comprising a bicyclic aromatic unit connected by an ester-containing bridge to another aromatic ring. Initial attempts to define a) the tissue selectivity of the hydrolytic metabolism and b) the molecular structural factors affecting the rate of enzymatic ester cleavage are presented. The data show that human and rat liver fractions were more active than human duodenal mucosa and human blood leukocytes at hydrolysing the compounds. The rank order of the compounds was, however, very similar in the different biological systems. Commercially available pig liver carboxyl esterase and cholesterol esterase both reasonably well predict the rank order in the tissue fractions.
TL;DR: The simultaneous enzymic liberation of acetyl groups from xylan and glucomannan clearly enhanced the action of xylan‐ and mannan‐degrading enzymes, thus increasing the hydrolysis yield significantly, however, none of the esterases was able to remove all acetyl substituents when acting alone and simultaneous action of two esterase was needed for complete deacetylation.
Abstract: Xylans and mannans contain different esterified substituents such as acetyl, feruloyl and p-coumaroyl side groups. The functions of hemicellulose-deacetylating esterases of Trichoderma reesei and Aspergillus oryzae are discussed in this paper. Both fungi produce multiple esterases and two different esterases were isolated from both T. reesei and A. oryzae. The enzymes differed significantly in their substrate specificities. Acetyl xylan esterase of T. reesei was highly active on polymeric xylan but was unable to remove acetyl substituents from glucomannan or phenolic substituents from wheat straw arabinoxylan. Another esterase, acetyl esterase from T. reesei, had activity only towards short oligomeric and monomeric acetates derived both from xylan and glucomannan. The acetyl glucomannan esterase of A. oryzae was most active towards polymeric glucomannan, but was also able to remove acetyl groups from xylan. The only esterase studied which was active against phenolic substituents in arabinoxylans was the feruloyl esterase from A. oryzae. Feruloyl esterase had the widest substrate specificity of the esterases studied. It was also able to act on acetyl groups both in xylan and in glucomannan. The simultaneous enzymic liberation of acetyl groups from xylan and glucomannan clearly enhanced the action of xylan- and mannan-degrading enzymes, thus increasing the hydrolysis yield significantly. However, none of the esterases was able to remove all acetyl substituents when acting alone and simultaneous action of two esterases was needed for complete deacetylation.
TL;DR: The esterase from Rhodosporidium toruloides has been used to catalyse the hydrolysis of a series of per-acetylated α-D-hexopyranoses and α- D- hexopyranosides, which have been selectively cleaved at the C-6 position to give the 6-OH derivatives.
Abstract: The esterase from Rhodosporidium toruloides has been used to catalyse the hydrolysis of a series of per-acetylated α-D-hexopyranoses and α-D-hexopyranosides. Per-acetylated glucose 4, mannose 6, N-acetylgalactosamine 8, galactose 10, methyl α-D-glucoside 12, methyl α-D-mannoside 14 and methyl α-D-galactoside 16 have been selectively cleaved at the C-6 position by the esterase to give the 6-OH derivatives 5, 7, 9, 11, 13, 15 and 17. Acid-catalysed rearrangement of acetates 5, 7, 13, 15, 11, 17 and 9 with 4→6 acetyl migration gives the corresponding 4-deprotected derivatives 22–28, respectively. Hydrolyses of β-D-glucose pentaacetate 20 and α-D-lactose octaacetate 21 have been attempted, but no hydrolyses have been observed. 1,2,3,6-Tetraacylated α-D-hexopyranoses 3 and 22, derivatives of N-acetylglucosamine and glucose respectively, and 2,3,6-triacetylated α-D-hexopyranosides 24 and 25, derivatives of glucose and mannose, respectively, have been hydrolysed by the esterase to the corresponding 4,6-dihydroxy acetates 29, 18, 30 and 31. Acylation of methyl α-D-glucopyranoside 32 catalysed by the esterase provides the C-6 monoacetate 33 and the C-3 monoacetate 34 in 4 and 5% yield, respectively. The sodium salts of N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose and mannose 6-sulfates 38–42, respectively, are prepared in two steps from the 6-deacetylated hexopyranoses 2, 5, 9, 11 and 7, respectively. The sodium salts of N-acetylglucosamine, glucose and mannose 4-sulfates 43–45, respectively, are prepared in two steps from the 4-deacetylated precursors 3, 22 and 26 which are obtained via acid catalysed 4→6 acyl migration of compounds 2, 5 and 7.
TL;DR: A recombinant esterase from Pseudomonas fluorescens (PFE) was produced from E. coli cultures and the enantioselectivity towards a series of racemic substrates was investigated.
TL;DR: Flow cytometry was used with different viability dyes to assess changes in cell structure and function induced by acetic acid (AA) in populations of Zygosaccharomyces bailii and SacCharomyces cerevisiae, and to detect changes in esterase activity, intracellular dye processing, and membrane integrity.
Abstract: Flow cytometry (FCM) was used with different viability dyes to assess changes in cell structure and function induced by acetic acid (AA) in populations of Zygosaccharomyces bailii (AA resistant) and Saccharomyces cerevisiae (AA sensitive). Kinetic changes in esterase activity, intracellular dye processing, and membrane integrity were monitored, and to detect those changes we used three assays involving fluorescein diacetate hydrolysis, FUN-1 processing, and propidium iodide exclusion, respectively. In S. cerevisiae, the decrease in the ability to process FUN-1 preceded the decrease in esterase activity, and there was loss of cell membrane integrity after incubation with AA. In Z. bailii, with higher AA concentrations, there was a similar decrease in the ability to process FUN-1, which also preceded the loss of cell membrane integrity. Changes in esterase activity in this yeast induced by AA treatment could not be monitored because the changes occurred independently of the presence of the acid. For control samples (untreated cells killed with 10% v/v of AA), the percentages of nonaltered cells as estimated by FCM and percentages of viable cells as estimated by colony forming unit (CFU) counts were identical. However, for cell samples treated for short periods with 3% (v/v) or less of AA, none of the dyes produced FCM results comparable to those produced by CFU counts.
TL;DR: It is proposed that molinate-induced inhibition of esterase activity in the Leydig cell could inhibit the mobilization of cholesterol esters required for testosterone biosynthesis.
TL;DR: The presence of the A2-B2 haplotype cannot be used to indicate the level of amplification or any particular degree of resistance in Culex pipiens mosquitoes, as the maps were almost identical in each strain.
Abstract: In Culex pipiens mosquitoes, A2 esterase alleles are co-amplified with B2 esterase alleles in response to selection with organophosphate insecticides. In this study the amplified A2 and B2 sequences were compared between twelve strains from four continents by restriction mapping. The restriction maps were almost identical in each strain throughout 22 kb surrounding the genes, suggesting that this represents a constant core sequence. A polymorphism was found in two strains collected from Egypt and Kenya in the mid 1980s. This polymorphism was present in all copies of the amplicon, which suggests that a mechanism of sequence homogenization was operating, i.e. concerted evolution. These two strains were almost certainly descendants from the same population and migration probably occurred along the River Nile. Although the maps were almost identical in each strain, dot blotting demonstrated that amplification levels differed by up to 13-fold between strains. Thus the presence of the A2-B2 haplotype cannot be used to indicate the level of amplification or any particular degree of resistance.
TL;DR: It is shown that this O-acetylation of sialic acids is due to the activity of an acetyl-coenzyme A:sialate-4-O-acetoltransferase (EC 2.3.1.44), which occurs together with sialyltransferase activity in Golgi-enriched membrane fractions of guinea-pig liver.
Abstract: Sialic acids from the liver and serum of guinea-pig are composed of N-acetylneuraminic acid (Neu5Ac; 85% and 61%, respectively), N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2; 10% and 32%, respectively) and N-glycolylneuraminic acid (Neu5Gc; 5% and 7%, respectively), besides traces of N-glycolyl-4-O-acetylneuraminic acid in serum. The analysis was carried out using thin-layer chromatography, high-performance liquid chromatography, electron impact ionization mass spectrometry, and different enzymes (sialidase, sialate esterase, and sialate-pyruvate lyase after hydrolysis and purification of the sialic acids by ion-exchange chromatography). We showed that this O-acetylation of sialic acids is due to the activity of an acetyl-coenzyme A:sialate-4-O-acetyltransferase (EC 2.3.1.44), which occurs together with sialyltransferase activity in Golgi-enriched membrane fractions of guinea-pig liver. The enzyme operates optimally at 30 degrees C in 70 mM potassium phosphate buffer at pH 6.7 and in the presence of 90 mM KCI with an apparent KM for AcCoA of 0.6 1microM and a Vmax of 20 pmol/mg protein x min. The enzyme is inhibited by coenzyme A in a mixed-competitive manner (Ki = 4.2 microM), as well as by parachloromercuribenzoate, MnCl2, saponin and Triton X-100.
TL;DR: The apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate and is a member of the GDSL family of serine esterases/lipases.
Abstract: Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.
TL;DR: Basic biochemical, immunological and molecular analysis suggests that the C. tritaeniorhynchus CtrEstβ1 gene is orthologous to the C quinquefasciatus amplified Est7beta;s, and the location of this gene within the genome predisposes it to amplification.
Abstract: Culex tritaeniorhynchus, the major vector of Japanese encephalitis in Sri Lanka, is resistant to organophosphorus insecticides, with a 10-fold resistance ratio at the LC50 for chlorpyrifos, and a high heterogenelty factor in the insect field population. The major mechanism of resistance in this species, as in the mosquito C. quinquefasciatus, is elevation of esterase activity. Basic biochemical, immunological and molecular analysis suggests that the C. tritaeniorhynchus CtrEstbeta1 gene is orthologous to the C. quinquefasciatus amplified Estbetas. The Estbeta2(1) antiserum cross-reacts strongly with CtrEstbeta1(1). Its corresponding cDNA, over the 545 base pairs sequenced, has approximately 84% identity with the various C. quinquefasciatus Estbetas. The gene is amplified in C. tritaeniorhynchus. Amplification of the same esterase in two independent species, along with multiple amplification events involving this esterase gene in C. quinquefasciatus suggests that the location of this gene within the genome predisposes it to amplification.