The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser(1β), is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.

MOLLA, GIANLUCA;ROSINI, ELENA;POLLEGIONI, LOREDANO;
2013-01-01

Abstract

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser(1β), is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.
2013
http://dx.doi.org/10.1042/BJ20121715
Amidohydrolases; chemistry/metabolism, Catalysis, Catalytic Domain, Cephalosporins; metabolism, Crystallography; X-Ray, Kinetics, Mutation, Penicillin Amidase; chemistry/genetics/metabolism, Protein Conformation, Pseudomonas; enzymology, Recombinant Proteins; chemistry/genetics/metabolism, Serine; metabolism, Substrate Specificity
E., Golden; R., Paterson; W. J., Tie; A., Anandan; G., Flematti; Molla, Gianluca; Rosini, Elena; Pollegioni, Loredano; A., Vrielink
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11383/1815523
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