4HSR

Crystal Structure of a class III engineered cephalosporin acylase

Summary for 4HSR

• Entry DOI: 10.2210/pdb4hsr/pdb
• Related: 4HST
• Descriptor: glutaryl-7-aminocephalosporanic acid acylase alpha chain, glutaryl-7-aminocephalosporanic acid acylase beta chain, 5,5-dihydroxy-L-norvaline, ... (4 entities in total)
• Functional Keywords: protein engineering, substrate specificity, transition state analogue, n-terminal hydrolase, hydrolase
• Biological source: Pseudomonas; More
• Total number of polymer chains: 2
• Total formula weight: 84333.37

Authors

Vrielink, A.,Golden, E.,Patterson, R.,Tie, W.J.,Anandan, A.,Flematti, G.,Molla, G.,Rosini, E.,Pollegioni, L. (deposition date: 2012-10-30, release date: 2013-02-27, Last modification date: 2024-02-28)

Primary citation

Golden, E.,Paterson, R.,Tie, W.J.,Anandan, A.,Flematti, G.,Molla, G.,Rosini, E.,Pollegioni, L.,Vrielink, A.
Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.
Biochem.J., 451:217-226, 2013

PubMed 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.

PubMed: 23373797
DOI: 10.1042/BJ20121715

Experimental method

X-RAY DIFFRACTION (2.13 Å)