Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263 (original) (raw)
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Ligand-induced conformational change in penicillin acylase
Journal of Molecular Biology, 1998
The enzyme penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) catalyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The enzyme is of great pharmaceutical importance, as the product 6-APA is the starting point for the synthesis of many semi-synthetic penicillin antibiotics. Studies have shown that the enzyme is speci®c for hydrolysis of phenylacetamide derivatives, but is more tolerant of features in the rest of the substrate. It is this property that has led to many other applications for the enzyme, and greater knowledge of the enzyme's structure and speci®city could facilitate engineering of the enzyme, enhancing its potential for chemical and industrial applications.
Protein Engineering Design and Selection, 2004
Penicillin acylase catalyses the condensation of Casubstituted phenylacetic acids with b-lactam nucleophiles, producing semi-synthetic b-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Ca-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, aF146Y, bF24A and aF146Y/bF24A, which all had a 2-to 10-fold higher affinity for Ca-substituted compounds than wildtype enzyme. In addition, bF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of aF146Y for (R)-a-methylphenylacetic acid can be explained by van der Waals interactions between aY146:OH and the Ca-substituent. The bF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-a-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the bF24A structure with other open conformations of penicillin acylase showed that bF24 has a fixed position, whereas aF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.
Structural Studies of Penicillin Acylase
Applied Biochemistry and Biotechnology, 2000
Penicillin acylases are used in the pharmaceutical industry for the preparation of antibiotics. The 3-D structure of Penicillin G acylase from Escherichia coli has been solved. Here, we present structural data that pertain to the unanswered questions that arose from the original strucutre. Specificity for the amide portion of substrate was probed by the structure determination of a range of complexes with substitutions around the phenylacetyl ring of the ligand. Altered substrate specificity mutations derived from an in vivo positive selection process have also been studied, revealing the structural consequences of mutation at position B71. Protein processing has been analyzed by the construction of site-directed mutants, which affect this reaction with two distinct phenotypes. Mutations that allow processing but yield inactive protein provide the structure of an ES complex with a true substrate, with implications for the enzymatic mechanism and stereospecificity of the reaction. Mutations that preclude processing have allowed the structure of the precursor, which includes the 54 amino acid linker region normally removed from between the A and B chains, to be visualized.
Journal of Molecular Biology, 2001
The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A Ê from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate speci®city and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a``virtual'' base.
Journal of Molecular Biology, 2001
The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A Ê from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate speci®city and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a``virtual'' base.
Biochemical Journal, 2003
Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl--leucine for growth Appl. Environ. Microbiol. 55, 2550-2555, are changed within one codon, replacing the B-chain residue Phe B(" with either Cys or Leu. Increases of up to a factor of ten in k cat \K m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K s . Values of k cat \K m for glutaryl--leucine are increased at least 100-fold. A decrease in k cat \K m for the Cys B(" mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure
Chembiochem, 2003
A three-dimensional model of the relatively unknown penicillin acylase from Alcaligenes faecalis (PA-AF) was built up by means of homology modeling based on three different crystal structures of penicillin acylase from various sources. An in silico selectivity study was performed to compare this homology model to the structure of the Escherichia coli enzyme (PA-EC) in order to find any selectivity differences between the two enzymes. The program GRID was applied in combination with the principal component analysis technique to identify the regions of the active sites where the PAs potentially engage different interactions with ligands. These differences were further analyzed and confirmed by molecular docking simulations. The PA-AF homology model provided the structural basis for the explanation of the different enantioselectivities of the enzymes previously demonstrated experimentally and reported in the literature. Different substrate selectivities were also predicted for PA-AF compared to PA-EC. Since no crystallographic data are available for PA-AF to date, the three-dimensional homology model represents a useful and efficient tool for fully exploiting this attractive and efficient biocatalyst, particularly in enantioselective acylations of amines.
Protein Science, 2015
Penicillin acylases are industrially important enzymes for the production of 6-APA, which is used extensively in the synthesis of secondary antibiotics. The enzyme translates into an inactive single chain precursor that subsequently gets processed by the removal of a spacer peptide connecting the chains of the mature active heterodimer. We have cloned the penicillin G acylase from Kluyvera citrophila (KcPGA) and prepared two mutants by site-directed mutagenesis. Replacement of N-terminal serine of the b-subunit with cysteine (Serb1Cys) resulted in a fully processed but inactive enzyme. The second mutant in which this serine is replaced by glycine (Serb1Gly) remained in the unprocessed and inactive form. The crystals of both mutants belonged to space group P1 with four molecules in the asymmetric unit. The three-dimensional structures of these mutants were refined at resolutions 2.8 and 2.5 Å , respectively. Comparison of these structures with similar structures of Escherichia coli PGA (EcPGA) revealed various conformational changes that lead to autocatalytic processing and consequent removal of the spacer peptide. The large displacements of residues such as Arg168 and Arg477 toward the Nterminal cleavage site of the spacer peptide or the conformational changes of Arg145 and Phe146 near the active site in these structures suggested probable steps in the processing dynamics. A comparison between the structures of the processed Serb1Cys mutant and that of the processed form of EcPGA showed conformational differences in residues Arga145, Phea146, and Pheb24 at the substrate binding pocket. Three conformational transitions of Arga145 and Phea146 residues were seen when processed and unprocessed forms of KcPGA were compared with the substrate bound structure of EcPGA. Structure mediation in activity difference between KcPGA and EcPGA toward acyl homoserine lactone (AHL) is elucidated.
Biochemical Journal, 1999
Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a k cat of 0.8 s −" and a K m of 10 µM at pH 7.5 and 20 mC. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s −" and a subsequent deacylation step with a rate constant of 0.81 s −" . Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to K S was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At