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 ability of lipases (triacylglycerol hydrolases, E.C. 3.1.1.3) to catalyze the hydrolysis of triacylglycerols has been known for many decades. However, since the early 1990s their importance in biotechnology has expanded significantly, due to their ability to catalyze reactions in nonconventional media such as organic solvents and to recognize various different esters as substrates. For example, lipases have been used to catalyze the transesterification of triacylglycerols and in the racemic resolution of various chiral esters. These special uses have driven a search for lipases able to catalyze novel reactions. Considering that typically such reactions are carried out under aggressive reactions conditions, such as the presence of organic solvents, it is necessary to find lipases that have high stability and activity under these conditions.The discovery of new enzymes through bioprospection, screening of metagenomic libraries, or molecular evolutionary approaches demands a practical and sensitive screening method capable of manipulating a large number of samples of either whole cells or crude enzyme extracts. The development of such methods for screening lipases is hampered by the physicochemical properties of lipids. Methods that rely on titrimetric or radiometric measurements have been used, but methods in the first group have low sensibility while those in the second group require expensive radiolabeled substrates. When a large number of samples are assayed, chromogenic or fluorogenic substrates are used. These substrates are esters from which the enzymatic hydrolysis liberates either a chromogenic or a fluorogenic agent that can be accompanied by UV/visible or fluorescence spectrophotometry, respectively. Note that fluorogenic substrates such as 4-methylumbelliferone (MUF)1 esters are more sensitive than 4-nitrophenyl esters. The use of MUF esters with quantification by fluorescence spectrophotometry to determine lipase and esterase activity has been described [6]. Here, we describe an image-based screening approach to quantify the activity of lipases using MUF-butyrate as the substrate. Images of the reactions in microtiter plates were captured with a charge-coupled device (CCD) camera.
        
Title: Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes Reiter B, Glieder A, Talker D, Schwab H Ref: Applied Microbiology & Biotechnology, 54:778, 2000 : PubMed
By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.
The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic-psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus, which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability.
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 ability of lipases (triacylglycerol hydrolases, E.C. 3.1.1.3) to catalyze the hydrolysis of triacylglycerols has been known for many decades. However, since the early 1990s their importance in biotechnology has expanded significantly, due to their ability to catalyze reactions in nonconventional media such as organic solvents and to recognize various different esters as substrates. For example, lipases have been used to catalyze the transesterification of triacylglycerols and in the racemic resolution of various chiral esters. These special uses have driven a search for lipases able to catalyze novel reactions. Considering that typically such reactions are carried out under aggressive reactions conditions, such as the presence of organic solvents, it is necessary to find lipases that have high stability and activity under these conditions.The discovery of new enzymes through bioprospection, screening of metagenomic libraries, or molecular evolutionary approaches demands a practical and sensitive screening method capable of manipulating a large number of samples of either whole cells or crude enzyme extracts. The development of such methods for screening lipases is hampered by the physicochemical properties of lipids. Methods that rely on titrimetric or radiometric measurements have been used, but methods in the first group have low sensibility while those in the second group require expensive radiolabeled substrates. When a large number of samples are assayed, chromogenic or fluorogenic substrates are used. These substrates are esters from which the enzymatic hydrolysis liberates either a chromogenic or a fluorogenic agent that can be accompanied by UV/visible or fluorescence spectrophotometry, respectively. Note that fluorogenic substrates such as 4-methylumbelliferone (MUF)1 esters are more sensitive than 4-nitrophenyl esters. The use of MUF esters with quantification by fluorescence spectrophotometry to determine lipase and esterase activity has been described [6]. Here, we describe an image-based screening approach to quantify the activity of lipases using MUF-butyrate as the substrate. Images of the reactions in microtiter plates were captured with a charge-coupled device (CCD) camera.
        
Title: Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes Reiter B, Glieder A, Talker D, Schwab H Ref: Applied Microbiology & Biotechnology, 54:778, 2000 : PubMed
By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.