Substrate Report for: 4-methylumbelliferyl-acetate

The carbohydrate esterase family 7 (CE7) members are acetyl esterases that possess unusual substrate specificity for cephalosporin C and 7-amino-cephalosporanic acid. This family containing the alpha/beta hydrolase fold has a distinctive substrate profile that allows it to carry out hydrolysis of esters containing diverse alcohol moieties while maintaining narrow specificity for an acetate ester. Here we investigate the structural basis of this preference for small acyl groups using the crystal structure of the thermostable Thermotoga maritima CE7 acetyl esterase (TmAcE) complexed with a non-cognate substrate analog. The structure determined at 1.86 A resolution provides direct evidence for the location of the largely hydrophobic and rigid substrate binding pocket in this family. Furthermore, a three-helix insertion domain near the catalytic machinery shapes the substrate binding site. The structure reveals two residues (Pro228 and Ile276) which constitute a hydrophobic rigid binding surface for the acyl group of the ester and thus restricts the size of the acyl group that be accommodated. In combination with previous literature on kinetic properties of the enzyme, our studies suggest that these residues determine the unique specificity of the TmAcE for short straight chain esters. The structure provides a template for focused attempts to engineer the CE7 enzymes for enhanced stability, selectivity or activity for biocatalytic applications.

The carbohydrate esterase family 7 (CE7) belonging to the alpha/beta hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical alpha/beta hydrolase fold. This element called the beta-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75A and 1.76A, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the beta-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability.

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database. Real-time polymerase chain reaction was used to determine the abundance of the CES2 and splice variant of human carboxylesterase 2 (CES2Delta(458-473)) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2Delta(458-473) transcript accounts for an average of 6% of total CES2 transcripts in colon tissue, and there is large interindividual variation in CES2 expression in both tumor and normal colon samples. The carboxylesterase activity of the colon samples was determined by 4-methylumbelliferyl acetate hydrolysis assays and nondenaturing polyacrylamide gel electrophoresis followed by activity staining. Significant, positive correlations were found between CES2 expression levels and both measures of carboxylesterase activity. We cloned and expressed the CES2Delta(458-473) protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2Delta(458-473) protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2Delta(458-473) protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2Delta(458-473) protein is an inactive splice variant of CES2 and that its transcript is spliced at a relatively constant rate in tumor and normal colon tissue.

This report deals with the purification, characterization, and a preliminary crystallographic study of a novel cold-active esterase (HaEst1) from Halocynthiibacter arcticus. Primary sequence analysis reveals that HaEst1 has a catalytic serine in G-x-S-x-G motif. The recombinant HaEst1 was cloned, expressed, and purified. SDS-PAGE and zymographic analysis were carried out to characterize the properties of HaEst1. A single crystal of HaEst1 was obtained in a solution containing 10% (w/v) PEG 8000/8% ethylene glycol, 0.1 M Hepes-NaOH, pH 7.5. Diffraction data were collected to 2.10 A resolution with P21 space group. The final Rmerge and Rp.i.m values were 7.6% and 3.5% for 50-2.10 A resolution. The unit cell parameters were a = 35.69 A, b = 91.21 A, c = 79.15 A, and beta = 96.9deg

The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a broad range of alcohols and is unique in its activity towards cephalosporin C. The CE7 fold contains a conserved N-terminal extension that distinguishes it from the canonical alpha/beta hydrolase fold. The hexameric quaternary structure indicates that the N-terminus may affect activity and specificity by controlling access of substrates to the buried active sites via an entrance tunnel. In this context, we characterized the catalytic parameters, conformation and thermal stability of two truncation variants lacking four and ten residues of the N-terminal region of the hyperthermostable Thermotoga maritima CE7 acetyl esterase (TmAcE). The truncations did not affect the secondary structure or the fold but modulated the oligomerization dynamics. A modest increase was observed in substrate specificity for acetylated xylose compared with acetylated glucose. A drastic reduction of ~30-40 degrees C in the optimum temperature for activity of the variants indicated lower thermal stability. The loss of hyperthermostability appears to be an indirect effect associated with an increase in the conformational flexibility of an otherwise rigid neighboring loop containing a catalytic triad residue. The results suggest that the N-terminal extension was evolutionarily selected to preserve the stability of the enzyme.

The carbohydrate esterase family 7 (CE7) members are acetyl esterases that possess unusual substrate specificity for cephalosporin C and 7-amino-cephalosporanic acid. This family containing the alpha/beta hydrolase fold has a distinctive substrate profile that allows it to carry out hydrolysis of esters containing diverse alcohol moieties while maintaining narrow specificity for an acetate ester. Here we investigate the structural basis of this preference for small acyl groups using the crystal structure of the thermostable Thermotoga maritima CE7 acetyl esterase (TmAcE) complexed with a non-cognate substrate analog. The structure determined at 1.86 A resolution provides direct evidence for the location of the largely hydrophobic and rigid substrate binding pocket in this family. Furthermore, a three-helix insertion domain near the catalytic machinery shapes the substrate binding site. The structure reveals two residues (Pro228 and Ile276) which constitute a hydrophobic rigid binding surface for the acyl group of the ester and thus restricts the size of the acyl group that be accommodated. In combination with previous literature on kinetic properties of the enzyme, our studies suggest that these residues determine the unique specificity of the TmAcE for short straight chain esters. The structure provides a template for focused attempts to engineer the CE7 enzymes for enhanced stability, selectivity or activity for biocatalytic applications.

The carbohydrate esterase family 7 (CE7) belonging to the alpha/beta hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical alpha/beta hydrolase fold. This element called the beta-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75A and 1.76A, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the beta-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability.

Abstract 1. Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure-metabolic activity relationship was examined. 2. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains. 3. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. 4. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. 5. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes. 6. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens. 7. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.

Carboxylesterases (CXEs) are widely distributed in plants, where they have been implicated in roles that include plant defense, plant development, and secondary metabolism. We have cloned, overexpressed, purified, and crystallized a carboxylesterase from the kiwifruit species Actinidia eriantha (AeCXE1). The structure of AeCXE1 was determined by X-ray crystallography at 1.4 A resolution. The crystal structure revealed that AeCXE1 is a member of the alpha/beta-hydrolase fold superfamily, most closely related structurally to the hormone-sensitive lipase subgroup. The active site of the enzyme, located in an 11 A deep hydrophobic gorge, contains the conserved catalytic triad residues Ser169, Asp276, and His306. Kinetic analysis using artificial ester substrates showed that the enzyme can hydrolyze a range of carboxylester substrates with acyl groups ranging from C2 to C16, with a preference for butyryl moieties. This preference was supported by the discovery of a three-carbon acyl adduct bound to the active site Ser169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with paraoxon (IC50 = 1.1 muM) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC50 = 9.2 muM). The structure of AeCXE1 with paraoxon bound was determined at 2.3 A resolution and revealed that the inhibitor binds covalently to the catalytic serine residue, with virtually no change in the structure of the enzyme. The structural information for AeCXE1 provides a basis for addressing the wider functional roles of carboxylesterases in plants.

The capacity of human, minipig, and rat skin and liver subcellular fractions to hydrolyze the anesthetic ester procaine was compared with carboxylesterase substrates 4-methylumbelliferyl-acetate, phenylvalerate, and para-nitrophenylacetate and the arylesterase substrate phenylacetate. Rates of procaine hydrolysis by minipig and human skin microsomal and cytosolic fractions were similar, with rat displaying higher activity. Loperamide inhibited procaine hydrolysis by human skin, suggesting involvement of human carboxylesterase hCE2. The esterase activity and inhibition profiles in the skin were similar for minipig and human, whereas rat had a higher capacity to metabolize esters and a different inhibition profile. Minipig and human liver and skin esterase activity was inhibited principally by paraoxon and bis-nitrophenyl phosphate, classical carboxylesterase inhibitors. Rat skin and liver esterase activity was inhibited additionally by phenylmethylsulfonyl fluoride and the arylesterase inhibitor mercuric chloride, indicating a different esterase profile. These results have highlighted the potential of skin to hydrolyze procaine following topical application, which possibly limits its pharmacological effect. Skin from minipig used as an animal model for assessing transdermal drug preparations had similar capacity to hydrolyze esters to human skin.

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database. Real-time polymerase chain reaction was used to determine the abundance of the CES2 and splice variant of human carboxylesterase 2 (CES2Delta(458-473)) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2Delta(458-473) transcript accounts for an average of 6% of total CES2 transcripts in colon tissue, and there is large interindividual variation in CES2 expression in both tumor and normal colon samples. The carboxylesterase activity of the colon samples was determined by 4-methylumbelliferyl acetate hydrolysis assays and nondenaturing polyacrylamide gel electrophoresis followed by activity staining. Significant, positive correlations were found between CES2 expression levels and both measures of carboxylesterase activity. We cloned and expressed the CES2Delta(458-473) protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2Delta(458-473) protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2Delta(458-473) protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2Delta(458-473) protein is an inactive splice variant of CES2 and that its transcript is spliced at a relatively constant rate in tumor and normal colon tissue.

S-Formylglutathione hydrolases (SFGHs) are highly conserved thioesterases present in prokaryotes and eukaryotes, and form part of the formaldehyde detoxification pathway, as well as functioning as xenobiotic-hydrolysing carboxyesterases. As defined by their sensitivity to covalent modification, SFGHs behave as cysteine hydrolases, being inactivated by thiol alkylating agents, while being insensitive to inhibition by organophosphates such as paraoxon. As such, the enzyme has been classified as an esterase D in animals, plants and microbes. While SFGHs do contain a conserved cysteine residue that has been implicated in catalysis, sequence analysis also reveals the classic catalytic triad of a serine hydrolase. Using a combination of selective protein modification and X-ray crystallography, AtSFGH from Arabidopsis thaliana has been shown to be a serine hydrolase rather than a cysteine hydrolase. Uniquely, the conserved reactive cysteine (Cys59) previously implicated in catalysis lies in close proximity to the serine hydrolase triad, serving a gate-keeping function in comprehensively regulating access to the active site. Thus, any covalent modification of Cys59 inhibited all hydrolase activities of the enzyme. When isolated from Escherichia coli, a major proportion of recombinant AtSFGH was recovered with the Cys59 forming a mixed disulfide with glutathione. Reversible disulfide formation with glutathione could be demonstrated to regulate hydrolase activity in vitro. The importance of Cys59 in regulating AtSFGH in planta was demonstrated in transient expression assays in Arabidopsis protoplasts. As determined by fluorescence microscopy, the Cys59Ser mutant enzyme was shown to rapidly hydrolyse 4-methylumbelliferyl acetate in paraoxon-treated cells, while the native enzyme was found to be inactive. Our results clarify the classification of AtSFGHs as hydrolases and suggest that the regulatory and conserved cysteine provides an unusual redox-sensitive regulation to an enzyme functioning in both primary and xenobiotic metabolism in prokaryotes and eukaryotes.

Twelve single nucleotide polymorphisms (SNPs) in the human CES2 gene, which encodes a carboxylesterase, hCE-2 [human carboxylesterase 2 (EC 3.1.1.1)], have been reported in the Japanese. In this report, we have examined functional alterations of three SNPs, a nonsynonymous SNP (100C>T, R34W), an SNP at the splice acceptor site in intron 8 (IVS8-2A>G), and one newly discovered nonsynonymous SNP (424G>A, V142M). For the two nonsynonymous SNPs, the corresponding variant cDNAs were expressed in COS-1 cells. Both the R34W and V142M variants showed little esterase activities toward the anticancer agent irinotecan and two typical carboxylesterase substrates, p-nitrophenol acetate and 4-methylumbelliferyl acetate, although increased levels of cDNA-mediated protein expression were observed by Western blotting as compared with the wild type. To investigate a possible splicing aberration in IVS8-2A>G, an in vitro splicing assay was utilized and transcripts derived from CES2 gene fragments of the wild type and IVS8-2A>G were compared. Sequence analysis of the cloned transcripts revealed that IVS8-2A>G yielded mostly aberrantly spliced transcripts, including a deleted exon or a 32-bp deletion proximal to the 5' end of exon 9, which resulted in truncated hCE-2 proteins. These results suggested that 100C>T (R34W), 424G>A (V142M), and IVS8-2A>G are functionally deficient SNPs.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of alpha-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against alpha-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.

A cDNA from Arabidopsis thaliana resembling S-formylglutathione hydrolase (SFGH), an enzyme with putative roles in formaldehyde detoxification in animals and microorganisms, has been cloned and expressed in Escherichia coli. The purified recombinant Arabidopsis enzyme (AtSFGH) was a dimer composed of 31-kDa subunits. Like SFGHs from other sources, AtSFGH had thioesterase activity toward S-formylglutathione and carboxyesterase activity toward 4-methylumbelliferyl acetate. Unlike other SFGHs, the enzyme from Arabidopsis actively hydrolyzed S-acetylglutathione. AtSFGH activity was inhibited by heavy metals and sulfhydryl alkylating agents, but was insensitive to serine hydrolase inhibitors, suggesting that the enzyme was a cysteine-dependent hydrolase. Although Atsfgh transcripts were determined in plants and cultures of Arabidopsis, the respective enzyme could not be detected in planta after the esterase activities present were resolved using isoelectric focusing. Instead, Arabidopsis contained several carboxyesterases active toward alpha-naphthyl acetate, which were all sensitive to inhibition by the serine hydrolase inhibitor paraoxon.

Downstream of flhA, the Paracoccus denitrificans gene encoding glutathione-dependent formaldehyde dehydrogenase, an open reading frame was identified and called fghA. The gene product of fghA showed appreciable similarity with human esterase D and with the deduced amino acid sequences of open reading frames found in Escherichia coli, Haemophilus influenzae, and Saccharomyces cerevisiae. Mutating fghA strongly reduced S-formylglutathione hydrolase activity. The mutant was unable to grow on methanol and methylamine, indicating that the enzyme is essential for methylotrophic growth. S-Formylglutathione hydrolase appears to be part of a formaldehyde detoxification pathway that is universal in nature.