Proline iminopeptidase gene from Xanthomonas campestris pv. citri

Alonso, Jorge; Garcı́a, José Luis

doi:10.1099/13500872-142-10-2951

Cited by 26 publications

(19 citation statements)

References 28 publications

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“…Cloning of the gene which encodes AEH and using it for overproduction would be worthwhile since expression in the natural hosts is low, varying from 0.1 to 2% of the total cellular protein (16,18,29,34; this study). In the past, effort was put into cloning an aehA gene, but this was unsuccessful (3,25).…”

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confidence: 99%

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

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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...

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confidence: 99%

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

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“…In addition, we were unable to detect proline aminopeptidase activity in the culture fluid. Proline aminopeptidases from bacteria have also been reported to be intracellular aminopeptidases (Albertson and Koomey 1993;Atlan et al 1994;Kitazono et al 1994b;Alonso and Garcia 1996;Yoshimoto et al 1999).…”

Section: Analysis Of the Papaorf Sequencementioning

confidence: 99%

“…Enzymes of this class appear to have a strict specificity for N-terminal proline residues (Kitazono et al 1994b). Such monomeric enzymes have been characterised from Bacillus coagulans (Kitazono et al 1992), Lactobacillus delbrueckii Gilbert et al 1994), Neisseria gonorrhoeae (Albertson and Koomey 1993), Serratia marcescens (Yoshimoto et al 1999;Ito et al 2000) and Xanthomonas campestris (Alonso and Garcia 1996;Medrano et al 1998). The second class of enzymes comprise large multimeric proteins that are not only capable of hydrolysing terminal prolines but have also been reported to be capable of hydrolysing N-terminal hydroxyproline residues.…”

Section: Introductionmentioning

confidence: 99%

Characterisation of Aspergillus niger prolyl aminopeptidase

et al. 2005

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We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.

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“…Escherichia coli HB101 harboring the plasmid pJJ185 [19] was grown in LB medium. All purification procedures were performed at 4°C.…”

Section: Protein Purificationmentioning

confidence: 99%

Crystallization and preliminary X‐ray diffraction analysis of proline iminopeptidase from Xanthomonas campestris pv. citri

et al. 1997

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Proline iminopeptidase from Xanthomonas campestris pv. citri, displaying no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapour diffusion method. Two different orthorhombic crystal forms (space group C222 and 1222) were obtained from a solution containing NaCl or polyethylene glycol monomethyl ether (MW 5000) as precipitating agent for the native and lanthanum-derivatized protein, respectively. Complete diffraction data sets have been collected up to 2.6 A (native) and

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Proline iminopeptidase gene from Xanthomonas campestris pv. citri

Cited by 26 publications

References 28 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

Characterisation of Aspergillus niger prolyl aminopeptidase

Crystallization and preliminary X‐ray diffraction analysis of proline iminopeptidase from Xanthomonas campestris pv. citri

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