Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely distributed among microorganisms, including bacteria, yeast and filamentous fungi. It is used on an industrial scale for the production of 6-aminopenicillanic acid, the starting material for the synthesis of semi-synthetic penicillins. Its in vivo role remains unclear, however, and the observation that expression of the Escherichia coli enzyme in vivo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mode. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains. The N-terminal serine of the B chain reacts with phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for the serine hydroxyl group. Modifying this serine to a cysteine inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme, indicating that this must be an important recognition site for cleavage. Here we report the crystal structure of penicillin acylase at 1.9 A resolution. Our analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own alpha-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.
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PMID:Penicillin acylase has a single-amino-acid catalytic centre. 781 45

Escherichia coli (muT, mutD, Leu-) cells transformed with plasmid pYKD59 harbouring the pac gene encoding penicillin acylase (PA) from Kluyvera citrophila ATCC 21285 were exposed to environmental conditions that made expression of this enzyme essential for growth. Under these conditions, spontaneous mutants were isolated that used adipyl-L-leucine as the sole source of L-leucine. DNA sequencing of the mutant pac genes identified a transversion mutation of thymine to guanine at position 1163. This mutation was located in the beta-subunit of the enzyme and resulted in conversion of Phe-360 to valine. The assignment of this mutation to the shift in substrate specificity was further confirmed by site-directed mutagenesis. Secondary-structure prediction of the region surrounding Phe-360 suggests that this mutation should not produce any significant structural change. The purified mutant acylase was able to hydrolyse adipyl-, glutaryl-, valeryl-, caproyl-, heptanoyl- and phenoxyacetyl-L-leucine at pH 5 with greater efficiency than the wild-type enzyme. However, the mutant enzyme was not able to hydrolyse glutaryl-7-aminocephalosporanic acid and had lost 90% and 50% of activity on penicillin G and phenylacetyl-L-leucine respectively. Nevertheless, mutant PA retained its original activity on 6-nitro-3-phenylacetamidobenzoate and p-nitrophenylphenylacetate, suggesting that the binding specificity of PA by the acyl and amine moieties of the substrate are not independent phenomena. The small differences observed between the c.d. spectra of the mutant enzyme recorded at pH 5 and 8 suggest the existence of different conformational states at the two pH values, but these differences were indistinguishable from those observed in the native enzyme and cannot be correlated with the shift in substrate specificity. Our results demonstrate that it is possible to change the specificity of PA by laboratory evolution and use it to identify the amino acids involved in substrate recognition. However, the synchronous participation of the alpha- and beta-subunits in the complex induced-fit-like mechanism of acylases suggests that, to obtain new enzymes for industrial application, the selection pressure should be specifically designed for the compound of interest.
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PMID:Changing the substrate specificity of penicillin G acylase from Kluyvera citrophila through selective pressure. 798 Apr 57

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.
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PMID:The isopenicillin-N acyltransferase of Penicillium chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene. 834

Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.
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PMID:Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification. 921 Mar 49

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, Km = 0.0042 mM) and colorimetric (6-nitro-3-phenylacetamidobenzoic acid, Km = 0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (Ki = 0.016 mM). The enzyme is partially inhibited at high benzylpenicillin concentrations but the triple SES complex formed still retains 43% of the maximal catalytic activity; the affinity of benzylpenicillin for the second substrate molecule binding site is much lower (K(S)' = 54 mM) than for the first one. Phenylmethylsulfonyl fluoride was shown to be a very effective irreversible inhibitor, completely inactivating the penicillin acylase from A. faecalis in a few minutes at micromolar concentrations; this compound was used for enzyme active site titration. The absolute values of the determined kinetic parameters for enzymatic hydrolysis of 6-nitro-3-phenylacetamidobenzoic acid (k(cat) = 95 s(-1) and k(cat)/Km = 2.1 x 10(-7) M(-1) s(-1)) and benzylpenicillin (k(cat) = 54 s(-1) and k(cat)/Km = 1.3 x 10(-7) M(-1) s(-1)) by penicillin acylase from A. faecalis were shown to be highest of all the enzymes of this family that have so far been studied.
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PMID:Kinetic study of penicillin acylase from Alcaligenes faecalis. 940 63

Penicillin acylase substrates suitable for colorimetric determination of the enzyme activity have been tested in this study. The kinetic parameters (Km and kcat) have been elucidated for the following nine substrates: six phenylacetic acid derivatives (p-nitroanilide, p-nitrophenyl ester, p-nitro-m-carboxyanilide, p-nitro-o-carboxyanilide, p-nitro-o-hydroxyanilide, m-nitro-p-carboxyanilide), two D-phenylglycine derivatives (p-nitroanilide, p-nitro-m-carboxyanilide), and also p-nitrophenyl ester of acetic acid (p-nitrophenyl acetate). With the exception of p-nitrophenyl acetate, all the compounds studied are highly specific chromogenic substrates for penicillin acylase, but their reactivity is very variable and kcat/Km values are in a range from 0.8.10(4) to 5.10(6) M(-1).sec(-1).
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PMID:Specific substrates for spectrophotometric determination of penicillin acylase activity. 979 83

The crystallization of a mutant precursor penicillin acylase [penicillin amidohydrolase (amidase) E.C. 3.5.1.11] from Escherichia coli W (ATCC 11105) using the hanging-drop method is reported. The crystals are in space group P1 with unit-cell parameters a = 51.04, b = 63.58, c = 71.17 A, alpha = 103.0, beta = 110.6, gamma = 105.3 degrees, with one molecule in the asymmetric unit, and diffract to 1. 8 A using synchrotron radiation.
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PMID:Crystallization of a precursor penicillin acylase from Escherichia coli. 1021 3

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.
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PMID:Enzymatic synthesis of beta-lactam antibiotics using penicillin-G acylase in frozen media. 1045 36

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.
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PMID:Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd. 1045 29

Isolation and characterization of a beta-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1. 11)-producing organism is reported. The test strain was isolated by an enrichment technique with a substrate other than penicillins. The isolated strain belongs to the genus Alcaligenes. Phenylacetic acid was found to be the inducer of penicillin amidase. The amidase has a broad substrate spectrum. It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate. Immobilized cells of Alcaligenes sp. were shown to act as a reversible enzyme.
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PMID:Beta-lactamase-free penicillin amidase from Alcaligenes sp.: isolation strategy, strain characteristics, and enzyme immobilization. 1048 31


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