Preparation and General Properties of Crystalline Penicillin Acylase from <i>Escherichia coli </i>ATCC 11 105

Kutzbach, Carl; Rauenbusch, Erich

doi:10.1515/bchm2.1974.355.1.45

Cited by 186 publications

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“…The values obtained from progress curves for the hydrolysis of NIPAB were in good agreement with the values obtained with the initial rate experiments and with data reported by Kutzbach and Rauenbusch (8) and Kasche et al (14). For phenylacetic acid, inhibition constants ranging from 0.05 to 5 mM have been reported (3,7,8,10). However, the K m and K i values for other phenylacetylated compounds are all in the range of 10 to 200 M (9,14), which indicates that the K i value of 70 M for phenylacetic acid obtained in these experiments is correct.…”

Section: Kinetics Of Nipab Hydrolysis and Phenylacetic Acid Inhibitionmentioning

confidence: 67%

“…Moreover, the classical steady-state method of measuring initial rates is prone to severe error because of the strong product inhibition by phenylacetic acid. Due to these complications, K m values for penicillin G and K i values for phenylacetic acid of penicillin acylase of E. coli have been reported, which differ by several orders of magnitude (3,(7)(8)(9)(10).…”

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confidence: 99%

“…As a chromogenic substrate we used 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB) (Fig. 1) and analogs thereof, of which the hydrolysis causes an increase in absorbance at 405 nm due to the liberation of 5-amino-2-nitrobenzoic acid (8). Using numerical integration of the relevant differential equations for product formation, accurate estimates for various invisible substrates could be obtained.…”

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confidence: 99%

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The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Alkema

Floris

Janssen

1999

Analytical Biochemistry

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Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K m values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower k cat /K m values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.

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Section: Kinetics Of Nipab Hydrolysis and Phenylacetic Acid Inhibitionmentioning

confidence: 67%

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confidence: 99%

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confidence: 99%

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The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Alkema

Floris

Janssen

1999

Analytical Biochemistry

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“…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.…”

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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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“…The involvement of Trp at the active sites of enzymes from Proteus rettgeri and from E. coli has been suggested from inactivation studies (14). The Ser reagent phenylmethanesulfonyl fluoride, which structurally resembles the side chain of benzylpenicillin, inactivated the enzyme from E. coli (11) and an enzyme from P. rettgeni (5). During the preparation of this article, there was a report that Ser-290 may act as a nucleophile in catalysis (26).…”

Section: Resultsmentioning

confidence: 99%

Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105

1992

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Penicillin G acylase from Escherichia coli ATCC 11105 is synthesized from its precursor polypeptide into a catalytically active heterodimer via a complex posttranslational processing pathway. Substitutions in the pair of aminoacyl residues at the cleavage site for processing the small and large subunits were made. Their processing phenotypes and penicillin G acylase activities were analyzed. By the introduction of a prolyl residue at either position, the processing of the small subunit was blocked without a change in enzymatic activity. Four other substitutions had no effect. At the site for processing the large subunit, four substitutions out of the seven examined blocked processing. In general, penicillin G acylase activity seemed to be proportional to the efficiency of the large-subunit-processing step. Ser-290 is an amino acid critical for processing and also for the enzymatic activity of penicillin G acylase. In the mutant pAATC, in which Ser-290 is mutated to Cys, the precursor is processed, but there is no detectable enzymatic activity. This suggests that there is a difference in the structural requirements for the processing pathway and for enzymatic activity. Recombination analysis of several mutants demonstrated that the small subunit can be processed only when the large subunit is processed first. Some site-directed mutants from which signal peptides were removed showed partial processing phenotypes and reduced enzymatic activities. Their expression showed that the prerequisite for penicillin G acylase activity is the efficient processing of the large subunit and that the maturation of the small subunit does not affect the enzymatic activity.Penicillin G acylase (PGA) is an enzyme that catalyzes the hydrolysis of benzylpenicillin to give 6-aminopenicillanic acid, an intermediate in the production of semisynthetic penicillin (28). PGA from Escherichia coli ATCC 11105 is a periplasmic enzyme that is composed of the small subunit and the large subunit. For PGA to reach its catalytically active dimer form in the periplasm, multistep proteolytic processing from a single polypeptide must be accomplished by removing a signal peptide (26 residues) and a spacer peptide (54 residues) (24, 25). The proteolytic cleavage sites are located between the Ala-235-Ala-236 and Thr-289-Ser-290 pairs (18). This maturation mechanism is peculiar to PGA as a bacterial enzyme, though many viral proteins and eucaryotic polypeptide hormones require activation by proteolytic cleavage processes (6,7). But the enzymes involved in each processing step and their locations within the cell have not yet been identified.In this report, site-specific mutations at the proteolytic cleavage sites of PGA were constructed in order to obtain the necessary information for cleavage activation, especially (i) the amino acids critical to PGA processing and PGA activity, (ii) the amino acid specificity of each cleavage step, and (iii) the order of the cleavage steps. Through the expression of various recombinantpga genes, the characteristics of each...

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Preparation and General Properties of Crystalline Penicillin Acylase from Escherichia coli ATCC 11 105

Cited by 186 publications

References 0 publications

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

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

Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105

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