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

Abstract

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...

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

“…Molecular Cloning-To clone aehA in the NcoI and HindIII site of pBAD/Myc-HisA, resulting in pBADAT, the NcoI restriction site was first removed from the gene cloned in pAT (4). This was accomplished by PCR using the sense primer 5Ј-GAACTGCCTGTGTCTATGGATATTT-TCCGGGGC-3Ј, the compatible reverse complement primer, and the QuikChange site-directed mutagenesis kit of Stratagene (La Jolla, CA), resulting in pATdelNco.…”

“…Earlier experiments with inhibitors already suggested the importance of a histidine for the catalytic activity of AEH (12). However, common serine hydrolase inhibitors such as phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, or Pefabloc SC showed no inhibition of AEH activity (4,12). On the other hand, inhibition was observed with the serine hydrolase inhibitor p-nitrophenyl-pЈ-guanidino-benzoate (p-NPGB), but the inhibition was incomplete, which left uncertainty about the catalytic role of a serine in AEH (4).…”

“…However, common serine hydrolase inhibitors such as phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, or Pefabloc SC showed no inhibition of AEH activity (4,12). On the other hand, inhibition was observed with the serine hydrolase inhibitor p-nitrophenyl-pЈ-guanidino-benzoate (p-NPGB), but the inhibition was incomplete, which left uncertainty about the catalytic role of a serine in AEH (4). In this study, we used active site labeling, site-directed mutagenesis, and sequence analysis to demonstrate that AEH is a member of a class of ␤-lactam antibiotic acylases that belongs to the ␣/␤-hydrolase fold family and possesses a classical catalytic triad of Ser, Asp, and His.…”

“…Remarkable features of these enzymes are the ability to accept charged substrates such as ␣-amino acid esters, the preference for esters over amides, and the low pH optimum (pH 6.2) (2,3). Despite these attractive properties, a gene encoding an ␣-amino acid ester hydrolase (AEH) 1 was only recently cloned and characterized (4). Thus far, all the known ␤-lactam antibiotic acylases, such as penicillin G acylase (5), penicillin V acylase (6), and cephalosporin acylase (7), belong to the Ntn-hydrolase family.…”

“…The N-terminal amino acid sequence of the AEH was determined; it revealed a signal sequence, but no N-terminally located Thr, Ser, or Cys, characteristic for members of the Ntnhydrolase family, was found. It was therefore postulated that the AEHs belong to a new class of ␤-lactam antibiotic acylases (4).…”