The structure at 1.6 Å resolution of the protein product of the At4g34215 gene from Arabidopsis thaliana (original) (raw)
Related papers
FEBS Letters
MekB from Pseudomonas veronii and CgHle from Corynebacterium glutamicum belong to the superfamily of a/b-hydrolase fold proteins. Based on sequence comparisons, they are annotated as homoserine transacetylases in popular databases like UNIPROT, PFAM or ESTHER. However, experimentally, MekB and CgHle were shown to be esterases that hydrolyse preferentially acetic acid esters. We describe the x-ray structures of these enzymes solved to high resolution. The overall structures confirm the close relatedness to experimentally validated homoserine acetyl transferases, but simultaneously the structures exclude the ability of MekB and CgHle to bind homoserine and acetyl-CoA. Insofar the MekB and CgHle structures suggest dividing the homoserine transacetylase family into subfamilies, namely genuine acetyl transferases and acetyl esterases with MekB and CgHle as constituting members of the latter.
Catalytic role of conserved HQGE motif in the CE6 carbohydrate esterase family
FEBS Letters, 2007
An acetylxylan esterase (R.44), belonging to the carbohydrate esterase family 6 (CE6), retrieved from bovine rumen metagenome was analyzed. Molecular modelling and site-directed mutagenesis indicated that the enzyme possesses a catalytic triad formed by Ser 14 , His 231 and Glu 152 . The catalytic Ser and His have been identified in highly conserved sequences GQSX and DXXH in the CE6 family, respectively, and the active-site glutamate was part of a highly conserved sequence HQGE. This motif is situated near to the so-called Block III in the CE6 family and its role in catalysis has not been identified so far.
Applied and Environmental Microbiology, 2008
Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene aes1 , encoding the acetyl esterase (Aes1) of Hypocrea jecorina , was identified by amino-terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino acid residues with the first 19 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the secreted enzyme were 37,088 Da and pH 5.89, respectively. No significant homology was found between the predicated Aes1 and carbohydrate esterases of known families, but putative aes1 orthologs were found in genomes of many fungi and bacteria that produce cell wall-degrading enzymes. The aes1 transcript levels were high when the fungal cells were induced with sophorose, cellulose, oat spelt xylan, lactose, and arabinose. The recombinant Aes1 produced by H. jecorina transformed with aes1 under the cellobiohydrolase I promoter displayed properties similar to th...
Unique Regulation of the Active site of the Serine Esterase S-Formylglutathione Hydrolase
Journal of Molecular Biology, 2006
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 xenobiotichydrolysing 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 Arabidospsis 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.
2007
Table of contents VII 3.2.4.3. Some insoluble lipolytic enzymes from Streptomyces coelicolor and Streptomyces avermitilis 3.2.4.4. Expression of some genes using the expression vector pET-23b 4. Discussion 4.1. Biochemical characterisation of Est A 4.1.1. Substrate specificity and Est A kinetics 4.1.2. Effects of temperature on Est A activity and stability 4.1.3. Effects of pH on Est A 4.1.4. Effect of metal ions and inhibitors 4.1.5. Enantioselectivity profile of Est A 4.1.6. HSL family conserved motifs 4.2. Site directed mutagenesis 4.3. Random mutagenesis 4.4. Biochemical characterisation of Est B 4.5. Other enzymes 4.5.1. Activity of the esterase from the gene locus SCO 3644 4.5.2. The enzyme produced from the gene locus SCO 1265 4.5.3. The insoluble enzymes 4.6. Future prespectives 5. Summary 6. Literature 7. Appendix A: The hydrolysis of some chiral compounds using Est A B: The hydrolysis of some chiral compounds using Est B. Acknowledgment 1.1.1. Why are enzymes of interest? Enzymes are central to every biochemical process within living cells. They are responsible for nutrient degradation, synthesis of biological macromolecules from simple precursors, DNA repair and replication etc. Simply, enzymes catalyze nearly all the metabolic reactions and in their absence the reactions will proceed at very slow rate, incompatible with living dynamics i.e. their activities are necessary to sustain life (Whitford 2005). Enzymes are interesting not only because of the aforementioned physiological roles, but also for their use in several other commercial applications. Enzymes have extraordinary catalytic power, often greater than that of synthetic or inorganic catalysts. They have a high degree of specificity for their substrates. They accelerate chemical reactions tremendously, and they function in aqueous solutions under very mild conditions of temperature and pH. All of the previous had encouraged the employment of enzymes in various commercial applications such as therapeutic, diagnostic and analytical reagents and as catalysts in different industries e.g. dairy, paper, cosmetics, etc (Schmid et al., 2001). Various enzymes have been used as diagnostic tools for many years ago (e.g. alkaline phosphatase and peroxidase). They are used for the detection and quantification of some medically significant metabolites in biological samples. Enzymes are also largely used as labels in enzyme immunoassay (EIA). Enzymatic preparations are ideal diagnostic reagents, because they are highly selective and it possesses catalytic efficiency. 1.1.2. Enzyme mechanism Many common reactions in the biochemistry require chemical events that are unfavourable in the cellular environment, such as the transient formation of unstably charged intermediate or the collision of two molecules in a precise orientation required for the reaction. An enzyme solves these problems through providing a special environment within which a reaction can occur more rapidly. Enzymes are usually specific as to the reaction they catalyze and the substrate they act upon. Shape, charge complementarities and hydrophilic/hydrophobic characters of the enzymes and substrates are responsible for this specificity (Nelson and Cox 2005). 1.2.1. Structure of esterases and lipases All of the known esterases and lipases are proteins. The polypeptide chains with any posttranslational modification constitute the primary structure of the protein. The local conformation that the polypeptide chain attains to keep itself unstrained is called the secondary structure. α-helices and the β-sheets are the two common secondary structural compenents for all the lipolytic enzymes. The polypeptide chain folds in a particular fashion to produce a three-dimensional product with a tertiary structure. Individual protein chains may sometimes group together to form a complex of two or more monomers, which are the quaternary structure (Nelson and Cox 2005), e.g. an extracellular carboxylesterase from the basidiomycete Pleurotus sapidus is composed of eight identical subunits (Zorn et al., 2005). The determination of the 3D structure of both esterases and lipases indicates that the bacterial esterases/lipases contain the characteristic α/β hydrolase fold. The α/β hydrolase fold is characteristic for the largest group of structurally related enzymes (esterases, lipases, hydrolases, proteases, etc) with diverse catalytic functions (the α/β hydrolase fold family). The central enzyme core is formed by β-sheets of eight strands (Fig 1.2). An enzyme catalyzed reaction is distinguished from other reactions by taking place within a definite pocket on the enzyme called the active site, which is a very small portion of the enzyme around 10 amino acid residues. The catalytic site of esterases/lipases is a serine protease-like catalytic triad consisting of the amino acids serine (nucleophile), histidine and aspartate or glutamate (acid); the nucleophilic serine is located in a highly conserved pentapeptide Gly-X-Ser-X-Gly and the aspartate or the glutamate residue is bounded through a hydrogen bond to the histidine (Fig 1.2). 1.2.2. Catalytic activity of lipolytic enzymes Esterases and lipases show various catalytic activities with different specificties. Some lipases show different rates against mono-, di-and triglycerides. Some esters act against either primary or secondary esters while others act nonspesifically. Some lipolytic enzymes show stereospecificity and/or regioselectivity (Jensen et al., 1983). Some lipolytic enzymes require other substances called cofactors to exert catalytic activity. Cofactors may be essential inorganic metal ions (e.g. Fe 2+ , Mg 2+ , Ca 2+ , Cu 2+ , etc) or coenzymes, which are complex organic or metallo-organic molecule (e.g. coenzyme A). The metal ion or the coenzyme is called prosthetic group, when it bounds tightly or covalently to the enzyme protein (Whitford 2005). There are several reports about dependence of esterases and lipases on metal ions e.g the activity of nine lipases from six different Staphylococcus species are Ca 2+ dependent (Rosenstein and Gotz 2000). Also some esterases/lipases have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed. This binding can serve to increase or decrease the enzyme's activity (depending on the molecule and enzyme), providing a means for feedback regulation, e.g. long chain acyl coenzyme A has an inhibitory effect on the activity of HSL in adipocytes (Hu et al., 2005). 1.2.5. Differences between esterases and lipases Esterases can be distinguished from lipases by the phenomenon of "Interfacial activation". Lipases act at the interface generated by a hydrophobic substrate in a hydrophilic aqueous medium, a sharp increase in lipase activity observed, when the substrate starts to form an emulsion thereby presenting to the enzyme an interfacial area (i.e. minimum substrate concentration is required for lipases to achieve high level of activity) (Fig 1.6). High to low High to low to zero Trigylcerides (long chain) Yes Don't obey Michael-Menten High Usually high 1.2.7. Applications of esterases and lipases Esterases and lipases are widely used as industrial enzymes, the industrial demand for both increased constantly over the last 20 years. In 2000 the market value of lipoyltic enzymes was US $ 90 millions which represent around 7% of the whole enzymes market value (Walsh 2004). Esterases are employed in reactions where chemo-or regioselectivity is required. Ferulic, sinapic, caffeic and coumaric acids, which are widely used in food, beverage and perfume industries, are produced from their esters with the help of esterases. Esterases are used in dairies and for production of fruit juices, wine, beer and alcohol. Polyurethanases and cholesterol esterases are widely used for the degradation of some man made pollutants, plastics, polyurethane, polyesters, etc (Bornscheuer 2002; Panda and Gowrishankar 2005). Lipases are used in fat hydrolysis or as a catalyst in synthetic organic chemistry where their regioselectivity and enantioselectivity are desired characteristics (Philip et al., 2002). Lipases can be widely used in organic chemicals processing, detergent formulations, synthesis of biosurfactants, the oleochemical industry, the dairy industry, the agrochemical industry, paper and pulp manufacture, nutrition, cosmetics and pharmaceutical processing. The major commercial application for hydrolytic lipases is their use in laundry detergents. In 1913 was the first trial to add a pancreatic extract to a detergent preparation, but the surfactants inactivated the pancreatic enzymes. Later in the 1970s suitable lipases for incorporation in detergents were identified. Detergent enzymes make about 32% of the total lipase sales. An estimated 1000 tons of lipases are added to approximately 13 billion tons of detergents produced each year (Sharma et al., 2001; Walsh 2004; Lorenz and Eck 2005). the production of pharmaceutical intermediates were reported e.g. taxol synthesis, throumboxane-A2-antagonist, acetylcholine esterase inhibitors, anti-cholesterol drugs, antiinfective drugs, Ca channel blocker drugs, K channel blocking drugs, anti-arrhythmic agents and antiviral agents (Bornscheuer 2002; Panda and Gowrishankar 2005). A lipase from Serratia marcescens catalyzes the synthesis of a key intermediate for "Diltiazem", a major coronary vasodilator. Lipases are used in synthesis of anti-hypertensive agents such as angiotensin converting enzyme (ACE) inhibitors (e.g. captopril, enalapril, ceranopril,
Structure, 2000
Background: The complex polysaccharide rhamnogalacturonan constitutes a major part of the hairy region of pectin. It can have different types of carbohydrate sidechains attached to the rhamnose residues in the backbone of alternating rhamnose and galacturonic acid residues; the galacturonic acid residues can be methylated or acetylated. Aspergillus aculeatus produces enzymes that are able to perform a synergistic degradation of rhamnogalacturonan. The deacetylation of the backbone by rhamnogalacturonan acetylesterase (RGAE) is an essential prerequisite for the subsequent action of the enzymes that cleave the glycosidic bonds.
The atomic-resolution structure of a novel bacterial esterase
Structure, 2000
Background: A novel bacterial esterase that cleaves esters on halogenated cyclic compounds has been isolated from an Alcaligenes species. This esterase 713 is encoded by a 1062 base pair gene. The presence of a leader sequence of 27 amino acids suggests that this enzyme is exported from the cytosol. Esterase 713 has been over-expressed in Agrobacterium without this leader sequence. Its amino acid sequence shows no significant homology to any known protein sequence. Results: The crystal structure of esterase 713 has been determined by multiple isomorphous replacement and refined to 1.1 Å resolution. The subunits of this dimeric enzyme comprise a single domain with an α/β hydrolase fold. The catalytic triad has been identified as Ser206-His298-Glu230. The acidic residue of the catalytic triad (Glu230) is located on the β6 strand of the α/β hydrolase fold, whereas most other α/β hydrolase enzymes have the acidic residue located on the β7 strand. The oxyanion hole is formed by the mainchain nitrogens of Cys71 and Gln207 as identified by the binding of a substrate analogue, (S)-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid. Cys71 forms a disulphide bond with the neighbouring Cys72. Conclusions: Despite negligible sequence homology, esterase 713 has structural similarities to a number of other esterases and lipases. Residues of the oxyanion hole were confirmed by structural comparison with Rhizomucor miehei lipase. It is proposed that completion of a functional active site requires the formation of the disulphide bond between adjacent residues Cys71 and Cys72 on export of the esterase into the oxidising environment of the periplasmic space.
Proteins, structure, function and bioinformatics, 2015
A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptido-glycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg 21 protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin.