Synthesis of β-lactam antibiotics containing α-aminophenylacetyl group in the acyl moiety catalyzed by d-(—)-Phenylglycyl-β-lactamide amidohydrolase

Blinkovsky, Alexander M.; Markaryan, A.

doi:10.1016/0141-0229(93)90173-y

Cited by 41 publications

(17 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, cephalexin is the preferred ␤-lactam antibiotic for AEH, as described for other AEHs as well (9,17,18,28), and the enzyme should therefore be designated a cephalexin acylase.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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 ␣-amino acid ester hydrolases, i.e., pH 6, compared to a pH of 7.5 to 8 for penicillin G acylases (20), and the lack of inhibition by phenylacetic acid (9), a side product from the hydrolysis of penicillin G, are also interesting properties for biocatalytic applications.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Polderman-Tijmes

Jekel

Vries

et al. 2002

Appl Environ Microbiol

View full text Add to dashboard Cite

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...

show abstract

“…However, cephalexin is the preferred ␤-lactam antibiotic for AEH, as described for other AEHs as well (9,17,18,28), and the enzyme should therefore be designated a cephalexin acylase.…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Polderman-Tijmes

Jekel

Vries

et al. 2002

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…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.…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases

Barends

Polderman-Tijmes

Jekel

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

␣-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 ...

show abstract

Cleavage and Formation of Amide Bonds

Hoople¹

1998

Biotechnology

View full text Add to dashboard Cite

Synthesis of β-lactam antibiotics containing α-aminophenylacetyl group in the acyl moiety catalyzed by d-(—)-Phenylglycyl-β-lactamide amidohydrolase

Cited by 41 publications

References 16 publications

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

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

The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases

Cleavage and Formation of Amide Bonds

Contact Info

Product

Resources

About