Α-amino Acid Ester Hydrolase Research Articles

A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases

Research involving α/β hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/β hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a ‘hit’ or a ‘miss,’ in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/β hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

Purification and characterization of Stenotrophomonas maltophilia-derived l-amino acid ester hydrolase for synthesizing dipeptide, isoleucyl-tryptophan

In the present study, we purified α-amino acid ester hydrolase (AEH) from cell-free extracts of the Stenotrophomonas maltophilia strain HS1. The approximately 70-kDa AEH from S. maltophilia HS1 (SmAEH) was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and was present as a tetramer in gel-filtration experiments. The activity of the SmAEH enzyme was then determined by monitoring the synthesis of the antihypertensive agent dipeptide isoleucyl-tryptophan (Ile-Trp) from isoleucyl methyl ester (Ile-OMe) and tryptophan (Trp). In these experiments, SmAEH had wide substrate specificity for acyl donors, such as Gly-OMe, β-Ala-OMe, Pro-OMe and Trp-OMe and Ile-OMe, and maximal activity were observed under conditions of pH 9.0 and 30°C. SmAEH also showed the greatest stability at pH 9.0, whereas its activity was reduced by 40% after 10-min incubation at approximately 50°C. In subsequent activity assays in the presence of various metal ions, Ag+ strongly inhibited enzyme activity. Finally, SmAEH activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that the protein is a serine protease.

Rational Design of Practically Important Enzymes

Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of genome sequencing and various modern approaches in protein engineering allow one to identify protein of interest and to improve the enzyme properties for a particular process. This review describes the results on development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharomyces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively), have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH) yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability. New generation of PseFDH preparations with the coenzyme specificity changed from NAD+ to NADP+ have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase (DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino acid replacements have been shown to exert an additive effect, improving both kinetic parameters and increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned. α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Structural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The structure of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and characterized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.

Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic efficiencies towards target reactions.

Changing the specificity of α-amino acid ester hydrolase toward para-hydroxyl cephalosporins synthesis by site-directed saturation mutagenesis

α-Amino acid ester hydrolases (AEHs) catalyze the synthesis of β-lactam antibiotics containing an α-amino group with decreased activity toward antibiotics with a p-hydroxyl group. The AEH gene from Xanthomonas rubrillineans was cloned and expressed in Escherichia coli. Based on the crystal structure of the AEH and cefprozil complex, 13 residues not directly involved in substrate recognition were mutated individually. The resulting ~1,300 mutants were screened for activity using cefprozil as a model product based on spectrophotometric assay in a 96-well format. Mutants with improved cefprozil synthetic activity revealed the particular importance of positions 87, 131 and 175 for specificity. The mutant V131S with the highest initial rates of synthesis toward three p-hydroxyl cephalosporins showed 23 %, 17 % and 64 % increase in maximum product accumulation of cefadroxil, cefprozil and cefatrizine, respectively.

Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of β-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes’ S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.

Gene Cloning and Characterization of α-Amino Acid Ester Acyl Transferase inEmpedobacter brevisATCC14234 andSphingobacterium siyangensisAJ2458

The gene encoding α-amino acid ester acyl transferase (AET), the enzyme that catalyzes the peptide-forming reaction from amino acid methyl esters and amino acids, was cloned from Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458 and expressed in Escherichia coli. This is the first report on the aet gene. It encodes a polypeptide composed of 616 (ATCC14234) and 619 (AJ2458) amino acids residues. The V(max) values of these recombinant enzymes during the catalysis of L-alanyl-L-glutamine formation from L-alanine methylester and L-glutamine were 1,010 U/mg (ATCC14234) and 1,154 U/mg (AJ2458). An amino acid sequence similarity search revealed 35% (ATCC14234) and 36% (AJ2458) identity with an α-amino acid ester hydrolase from Acetobacter pasteurianus, which contains an active-site serine in the consensus serine enzyme motif, GxSYxG. In the deduced amino acid sequences of AET from both bacteria, the GxSYxG motif was conserved, suggesting that AET is a serine enzyme.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the α/β-hydrolase fold superfamily

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase (GCA) isolated from Bacillus laterosporus J1, has been conventionally grouped as the only member of class V GCA, although its amino acid sequence shares less than 10% identity with members of other classes of GCA. Instead, it shows higher sequence similarities with Rhodococcus sp. strain MB1 cocaine esterase ( RhCocE) and Acetobacter turbidans α-amino acid ester hydrolase ( AtAEH), members of the α/β-hydrolase fold superfamily. Homology modeling and secondary structure prediction indicate that the N-terminal region of J1 acylase has an α/β-hydrolase folding pattern. The catalytic triads in RhCocE and AtAEH were identified in J1 acylase as S125, D264 and H309. Mutations to alanine at these positions were found to completely inactivate the enzyme. These results suggest that J1 acylase is a member of the α/β-hydrolase fold superfamily with a serine–histidine–aspartate catalytic triad.

Identification of the Catalytic Residues of α-Amino Acid Ester Hydrolase from Acetobacter turbidans by Labeling and Site-directed Mutagenesis

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.

Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding an α-Amino Acid Ester Hydrolase from Acetobacter turbidans

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the alpha-amino acid ester hydrolase is a beta-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The alpha-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of beta-lactam antibiotic acylases.

Stabilization of a tetrameric enzyme (α-amino acid ester hydrolase from Acetobacter turbidans) enables a very improved performance of ampicillin synthesis

The stabilized derivative of the enzyme α-amino acid ester hydrolase from Acetobacter turbidans has been found to be very adequate as biocatalyst of the synthesis of the very relevant antibiotic ampicillin. This enzyme resulted much more adequate than the Penicillin G Acylase (PGA) from Escherichia coli (the most used enzyme). The stabilization of the enzyme was required because under optimal conditions (absence of phosphate and 40% of MeOH), no-stabilized derivatives or soluble enzyme from A. turbidans become very rapidly inactivated. Under these conditions, this new stabilized derivative exhibited a very high selectivity for the transferase activity compared to the esterase one, as well as a very low hydrolytic activity towards the antibiotic. Moreover, this new biocatalyst did not recognize l-phenylglycine as substrate in the synthetic process. By using the racemic mixture of d/l phenylglycine methyl ester, 85% of the d-ester could be transformed to ampicillin. In contrast, the enzyme from E. coli exhibited a high hydrolytic activity for the ampicillin yielding low synthetic yields. This enzyme also resulted much less enantioselective producing both isomers of the antibiotic.

Importance of enzyme and solvent physical properties for the biocompatibility relationship of α-amino acid ester hydrolase

The enzyme α-amino acid ester hydrolase was used with a variety of organic solvents to further explore the relationships between biocompatibility and solvent physical properties. Biocompatibility was shown to be system-specific; that is, the inflection point of the characteristic log P trend (approximately 1.5) was specific to the enzyme employed, while the biocompatibility of solvents that were exceptions to the trend was dependent on the extent of agitation. In addition, phase toxic solvents all possessed a high interfacial tension with water. Finally, while the source of the enzyme may be significant, the extent of purification does not appear to be a factor in biocompatibility.

Studies on .ALPHA.-amino acid ester hydrolase of Xanthomonas citri. I. Purification of .ALPHA.-amino acid ester hydrolase from Xanthomonas citri.

α-Amino acid ester hydrolase, which can synthesize various kinds of semi-synthetic cephalosporins from 7-aminocephem compounds and α-amino acid esters, was purified to homogeneity from the cell-free extract of Xanthomonas citri IFO 3835 by ion-exchange chromatography and gel filtration. The purified enzyme migrated as a single band on disc gel electrophoresis and sedimented as a single symmetric peak on ultracentrifugation (s20, w = 11.8S). It showed a UV absorption maximum at 280 nm at pH 7.0 and had an isoelectric point at pH 7.8. Cysteine and cystine were not found in its molecule. Carbohydrate was not substantially detected. The molecular weight was estimated to be 270,000-280,000 by ultracentrifugation and gel filtration. The enzyme dissociated into subunits with an identical molecular weight of 72,000 in the presence of sodium dodecyl sulfate or guanidine · HCl.

Studies on .ALPHA.-amino acid ester hydrolase of Xanthomonas citri. III. Kinetics of acyl transfer by .CHI.-amino acid ester hydrolase from Xanthomonas citri.

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (ko) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (KM), and plots of l/ko against the initial concentration of a nucleophile (no) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.

Studies on .ALPHA.-amino acid ester hydrolase of Xanthomonas citri. II. Substrate specificity of .ALPHA.-amino acid ester hydrolase from Xanthomonas citri.

Enzymological properties of the purified α-amino acid ester hydrolase from Xanthomonas citri IFO 3835 were studied. The enzyme catalyzed both hydrolysis of α-amino acid esters (e.g. D-α-phenylglycine methyl ester (D-PG-OMe)) and transfer of the acyl group from the amino acid esters to amine nucleophiles (e.g. 7-aminocephalosporanic acid, 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and 6-aminopenicillanic acid). The substrate specificity of the enzyme in hydrolysis was parallel with that in transfer. The enzyme required a free amino group on the α-carbon of the donor ester, but did not exhibit absolute stereospecificity. α-Amino acid derivatives having an acid-amide bond were hydrolyzed at much lower rates than the corresponding ester derivatives. The optimum pH for both hydrolysis and transfer was 6.4, and the optimum temperature, 35°C. The enzyme was inactivated completely within 15 min at pH 6.0 and 50°C. FeSO4, CuSO4, and HgCl2 inhibited the enzyme. At pH 6.4 and 30&C, the Michaelis constants were estimated to be 8.26mM for D-PG-OMe and 2.99mM for cephalexin. The apparent Michaelis constant for 7-ADCA was estimated to be 5.08 mm, With D-PG-OMe as an acyl donor, the molecular activity (ko) for hydrolysis was calculated to be 1.11×104 sec-1.

Enzymatic synthesis of amoxicillin by the cell-bound .ALPHA.-amino acid ester hydrolase of Xanthomonas citri.

Whole cells of Xanthomonas citri K24, a penicillinase-deficient α-amino acid ester hydrolase producer, were effectively used for synthesis of amoxicillin from d-α-(p-hydroxyphenyl)-glycine methyl ester (HPGME) and 6-aminopenicillanic acid (6-APA). The yield of amoxicillin from 6-APA was increased by reducing the ionic strength of the reaction mixture and by adding 2-butanol to the reaction mixture. Increasing the HPGME/6-APA ratio above 1:1 raised the yield of amoxicillin. The optimum pH for the synthetic reaction was between 6 and 7, and the optimum temperature, 20°C. The addition of 2-butanol repressed the enzymatic hydrolysis of HPGME, and enhanced the acylation of 6-APA. More than 90% of the added 6-APA was converted into amoxicillin under the optimum conditions.

Substrate specificity of an alpha-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325.

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.