Alpha-amino Acid Ester Hydrolase Research Articles

Recombinant alpha-amino ester acid hydrolase from Xanthomonas rubrilineans VKPM B-9915 is a highly efficient biocatalyst of cephalexin synthesis

Recombinant, as well as native alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 (XrAEH, EC 3.1.1.43), was tested for synthesis of amino-beta-lactam antibiotic cephalexin. It was shown that the recombinant enzyme r-XrAEH produced by Escherichia coli VKPM B-11246 is more efficient in comparison with the native enzyme wt-XrAEH prepared from mutant strain Xanthomonas rubrilineans VKPM B-9915. When r-XrAEH was used as a biocatalyst, addition of ethylene glycol (33 vol %) to the reaction medium improved the yield from 70 to 95%. During synthesis of cephalexin under optimal conditions in the case of the native enzyme wt-XrAEH the cephalexin yield was 85%, in contrast to r-XrAEH where it was 95%. Furthermore, unlike native wt-XrAEH enzymes, preparations of recombinant r-XrAEH do not possess beta-lactamase side activity.

3D Structure Modeling of Alpha-Amino Acid Ester Hydrolase from Xanthomonas rubrilineans

Alpha-amino acid ester hydrolase (EC 3.1.1.43, AEH) is a promising biocatalyst for the production of semi-synthetic β-lactam antibiotics, penicillins and cephalosporins. The AEH gene from Xanthomonas rubrilineans (XrAEH) was recently cloned in this laboratory. The three-dimensional structure of XrAEH was simulated using the homology modeling method for rational design experiments. The analysis of the active site was performed, and its structure was specified. The key amino acid residues in the active site - the catalytic triad (Ser175, His341 and Asp308), oxyanion hole (Tyr83 and Tyr176), and carboxylate cluster (carboxylate groups of Asp209, Glu310 and Asp311) - were identified. It was shown that the optimal configuration of residues in the active site occurs with a negative net charge -1 in the carboxylate cluster. Docking of different substrates in the AEH active site was carried out, which allowed us to obtain structures of XrAEH complexes with the ampicillin, amoxicillin, cephalexin, D-phenylglycine, and 4-hydroxy-D-phenylglycine methyl ester. Modeling of XrAEH enzyme complexes with various substrates was used to show the structures for whose synthesis this enzyme will show the highest efficiency.

A New Binding Site Involving the C-terminal Domain to Design Specific Inhibitors of PepX

PepX is a X-prolyl dipeptidyl aminopeptidase of S15 family that cleaves dipeptides from the N-terminus of polypeptides having a proline or alanine residue at the second position. Involved in bacterial nutrition and in peptide maturation, this serine exopeptidase, counterpart of the mammalian DDP-4, has been proposed to play a role in pathogenicity for Streptococci and to be a promising target against trypanosomes. Searching for specific inhibitors, we undertook docking simulations on the whole surface of PepX from Lactococcus lactis, type example of the S15 family, which revealed a new putative binding site in connection with the active site and involving the C-terminal domain. Accordingly to the results of the computations, we synthesized two peptidomimetics of low molecular weight: the valinephenylpiperazine and the valine-isopropylpiperazine that can accommodate to this putative binding site. Experiments revealed that the valine-phenylpiperazine was an uncompetitive inhibitor whereas the valine-isopropylpiperazine showed to be an activator of the enzyme activity. The valine-phenylpiperazine is interacting with ASN 379, GLU 383, GLU 474, residues in connection with the specificity and active sites, and with the residues from the C-terminal domain LEU 693, GLU 710 and GLN 712. These results point out a role of the C-terminal domain in controlling access to the active site of enzymes of the S15 family, like PepX, the cocaine esterase or the alpha-amino acid ester hydrolase, and could have applications in human health giving new perspectives to struggle against streptococci or trypanosomes by designing inhibitors specific to the S15 family of enzymes.

Acetobacter turbidans α-Amino Acid Ester Hydrolase: HOW A SINGLE MUTATION IMPROVES AN ANTIBIOTIC-PRODUCING ENZYME

The alpha-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of beta-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which D-phenylglycine binds, and an additional space that is large enough to accommodate the beta-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr(112), which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr(112) side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the beta-lactam binding site, this could explain the increased beta-lactam transferase activity of this mutant.

Acetobacter turbidans alpha-amino acid ester hydrolase: merohedral twinning in P21 obscured by pseudo-translational NCS.

The structure elucidation of the alpha-amino acid ester hydrolase from Acetobacter turbidans by molecular replacement is described. In the monoclinic crystal, the molecules are related by both rotational and pseudo-crystallographic translational NCS (non-crystallographic symmetry). Refinement of the structure converged at unacceptably high R factors. After re-evaluation of the data, it was found that the crystal was merohedrally twinned, with a high twinning fraction. It is shown that the pseudo-crystallographic NCS causes aberrant behaviour of conventional twinning indicators, which explains why the twinning was only realized at the refinement stage.

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.

Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions.

The importance of the stabilization of the quaternary structure of multimeric enzymes has been illustrated using a model reaction with great industrial relevance: the enzymatic synthesis of ampicillin from 6-amino penicillanic acid (6APA) and phenylglycine methyl ester (PGM) catalyzed by the tetrameric enzyme alpha-amino acid ester hydrolase from Acetobacter turbidans. The stabilization of the multimeric structure of the enzyme was achieved by multi-subunit immobilization of the enzyme followed by its further solid-phase chemical intersubunit cross-linking with polyfunctional macromolecules (dextran-aldehyde). This stabilized derivative has permitted the study of the reaction under conditions where nonstabilized enzyme molecules tended to dissociate (e.g., absence of phosphate ions). Synthetic yields improved from around 65%, under conditions where the nonstabilized derivative was stable, to around 85% in conditions where only the stabilized derivative could be utilized (40% methanol and absence of phosphate ions). When using high concentrations of PGM, a significant worsening of the reaction performance was detected with a significant decrease in the yields (below 55%, using 50 mM 6APA and PGM). This problem has been sorted out by using a fed-batch reaction system. By addition of PGM continuously to the reaction mixture (to maintain the concentration between 0.5 and 3 mM), 95% of 6-APA could be transformed to antibiotic (47.5 mM) by only using a 20% excess of acylating ester.

Enhancement effect of water activity on enzymatic synthesis of cephalexin

The effect of water activity (a(w)) of the reaction medium on the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenylglycine methyl ester (PGM) was investigated using the alpha-amino acid ester hydrolase enzyme from Xanthomonas citri. It was found that the synthetic activity of the enzyme and the conversion yield were markedly improved when the a(w) of the reaction medium was lowered to about 0.97. The water activity depressing agents evaluated were glycerol, sucrose, and sorbitol, and the conversion yields were improved up to 170% with 15% glycerol, 230% with 30% sucrose, and 270% with 20% sorbitol, respectively. The extent of favorable effect of a(w) on the conversion yield was not the same among the a(w) depressors, probably due to other unknown interactions between the enzyme and depressors. However, optimal a(w) values corresponding to the maximum conversion yield coincided for all a(w) depressors used. The conversion yield of CEX showed an increasing trend with increasing a(w) up to the optimal a(w) value (0.96-0.97) which corresponds to the maximum conversion yield and a decreasing trend beyond the optimal a(w). There appears to be a delicate balance between the hydrolytic reaction of PGM and synthetic reaction of CEX. The increasing a(w)-[E . PGM] complex and the branched reaction pathway fluxes from [E . PGM] to PG (D-alpha-phenyl glycine) and CEX are balanced in such a way that the maximum CEX conversion yield is obtained at a(w) value of 0.96-0.97. The a(w) depressors stabilized the enzyme somewhat, but this positive effect was considered to be only a minor contribution to the substantial yield enhancement. The a(w) depressor effect on viscosity and in turn the mass transfer rate limitation was ruled out since the change in conversion due to the viscosity change was found to be insignificant.

Isolation of dealanylalahopcin, a new amino acid, and its biological activity.

A new amino acid, dealanylalahopcin, was isolated from a culture filtrate of Streptomyces albulus subsp. ochragerus; it was also formed by the enzymatic hydrolysis of alahopcin using microbial alpha-amino acid ester hydrolase. The amino acid was obtained as colorless needles and its molecular formula is C6H10N2O5. It showed very weak antibacterial activity against Gram-positive and Gram-negative bacteria, and weak inhibitory activity against the collagen prolylhydroxylase.