␣-Amino acid ester hydrolases (AEHs) catalyze the hydrolysis and synthesis of esters and amides with an ␣-amino group. As such, they can synthesize ␤-lactam antibiotics from acyl compounds and ␤-lactam nuclei obtained from the hydrolysis of natural antibiotics. This article describes the gene sequence and the 1.9-Å resolution crystal structure of the AEH from Xanthomonas citri. The enzyme consists of an ␣/␤-hydrolase fold domain, a helical cap domain, and a jellyroll ␤-domain. Structural homology was observed to the Rhodococcus cocaine esterase, indicating that both enzymes belong to the same class of bacterial hydrolases. Docking of a ␤-lactam antibiotic in the active site explains the substrate specificity, specifically the necessity of an ␣-amino group on the substrate, and explains the low specificity toward the ␤-lactam nucleus.␤-Lactam antibiotics form a large family of widely applied antibacterials. Most of them are derived from a handful of naturally occurring antibiotics like penicillin G, penicillin V, and cephalosporin C by replacing their acyl groups with synthetic ones. Initially, this was achieved by chemical means but at present, enzymatic methods are preferred (1). A well known enzyme used for these conversions is penicillin acylase (EC 3.5.1.11) from Escherichia coli. This enzyme is used both for the production of the ␤-lactam nucleus 6-aminopenicillanic acid (6-APA) 1 by cleaving off phenylacetic acid from penicillin G and for the coupling of new acyl groups to 6-APA or other ␤-lactam nuclei. Penicillin acylase is, however strongly inhibited by its product phenylacetic acid (2), which must therefore be removed before coupling of a new acyl group to the ␤-lactam nucleus can take place. In addition, ␤-lactam nuclei are not very stable at the alkaline pH optimum of penicillin acylase.By contrast, ␣-amino acid ester hydrolases (AEHs) do not have these disadvantages. These enzymes catalyze the hydrolysis and synthesis of esters and amides of ␣-amino acids exclusively, and do not attack the amide bond of a ␤-lactam. They can be used to acylate a ␤-lactam using an ester as acyl donor, as shown in Fig. 1. Because the AEHs require an ␣-amino group on the substrate, they are not inhibited by phenylacetic acid (3). Together with their ability to accept various ␤-lactam nuclei without cleaving them, this makes them suitable for generating widely used antibiotics such as ampicillin, amoxicillin, and the cephalosporins cephadroxil and cephalexin. The slightly acidic pH optimum of AEH, which is beneficial for ␤-lactam stability, is another advantage of AEHs for biocatalytic applications, as is their stereospecificity toward the acyl donor (4).One of the first AEHs that was isolated and characterized is the enzyme from Xanthomonas citri (5-11). This enzyme was found to be a homotetramer with subunits of 72 kDa (6). Kinetic studies indicated the occurrence of an acyl-enzyme intermediate in the hydrolysis and acylation reactions of ␤-lactam antibiotics (3,7,12).This article reports the gene and 1.9-Å resolution ...

The ␣-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing ␤-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified ␣-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 ␣-amino acid ester hydrolase is a ␤-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 ␣-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of ␤-lactam antibiotic acylases.In the search for microorganisms to be used in the biocatalytic production of semisynthetic antibiotics, Acetobacter turbidans ATCC 9325 was first described in 1972 by Takahashi et al. (35) as an organism able to synthesize cephalosporins. Since only ␣-amino acid derivatives could act as substrates and due to the preference for esters over amides, the enzyme involved was named ␣-amino acid ester hydrolase (AEH) (34).A similar AEH (EC 3.1.1.43) activity has been described for several other organisms. These enzymes catalyze the transfer of the acyl group from ␣-amino acid esters to amine nucleophiles such as 7-aminocephem and 6-penam compounds (synthesis) or to water (hydrolysis) (Fig. 1). Presumably, an acylenzyme intermediate is involved in this transfer reaction (9, 34). These AEHs show promising properties for the industrial enzymatic production of semisynthetic ␤-lactam antibiotics. Due to the preference for esters, it is conceivable that higher product (amide) accumulation can be reached in synthesis reactions using these enzymes than with the Escherichia coli penicillin G acylase (EC 3.5.1.11) (15,28,34). Moreover, the enzyme of A. turbidans showed high selectivity toward the D-form of phenylglycine methyl ester (D-PGM, the activated acyl donor). This enables an ampicillin synthesis starting from a racemic mixture of acyl donors, which is not feasible with the E. coli penicillin acylase (14). In contrast to penicillin acylase from E. coli, it was claimed that the AEHs are able to accept charged substrates (9). The low pH optimum of the ␣-ami...

The ␣-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in ␤-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 ␣-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a 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 ␣-amino acid ester hydrolase has an ␣/␤-hydrolase fold structure with a catalytic triad of Ser 205 , Asp 338 , and His 370 . This distinguishes the ␣-amino acid ester hydrolase from the Ntnhydrolase family of ␤-lactam antibiotic acylases.

The ␣-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of ␤-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 ␤-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 ␤-lactam binding site, this could explain the increased ␤-lactam transferase activity of this mutant.Thirty years ago, several bacterial strains, such as Acetobacter turbidans and Xanthomonas citri, were identified that were able to efficiently produce semi-synthetic ␤-lactam antibiotics from ␤-lactam nuclei produced by fermentation, and synthetic acyl compounds with an ␣-amino group (1). Important antibiotics with such acyl chains include cephalexin, cephadroxil, ampicillin, and amoxicillin. Given the difficulties in preparing such antibiotics by chemical means (2), much effort has been put into harnessing the ␤-lactam antibiotic synthesizing activity of these bacteria for application in the industrial production of antibiotics. It appeared that this activity originated from enzymes preferentially hydrolyzing esters of ␣-amino acids, the ␣-amino acid ester hydrolases (AEHs) 2 (3). Because of its potential usefulness in antibiotic synthesis, the AEH from A. turbidans has been studied extensively, and it was the first of its family for which the gene was cloned and overexpressed (4). The sequence showed a GXSYXG active site motif (4), which is characteristic of serine hydrolases of the X-prolyl dipeptidyl aminopeptidase family (5). Labeling studies with a suicide inhibitor, sequence alignments, and site-directed mutagenesis identified a catalytic triad of Ser 205 , Asp 338 , and His 370 in what was proposed to be a catalytic domain with an ␣/␤-hydrolase fold (6).Recently, the crystal structure of the X. citri AEH was solved (7). This enzyme shares 63% sequence identity with the A. turbidans AEH. The structure showed a tetrameric arrangement of monomers consisting of three domains each: an ␣/␤-hydrolase domain at the N terminus, a helical cap domain, and a C-terminal jellyroll fold domain. The active site indeed contained a Ser-His-Asp catalytic triad, the constituents of which were found in their canonical positions in the ␣/␤-hydrolase domain. Furthermore, a putative oxyanion hole was found i...