title: Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.
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.
authors: School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
authors: Emily
authors: E
authors: Golden
authors: Rachel
authors: R
authors: Paterson
authors: Wan Jun
authors: WJ
authors: Tie
authors: Anandhi
authors: A
authors: Anandan
authors: Gavin
authors: G
authors: Flematti
authors: Gianluca
authors: G
authors: Molla
authors: Elena
authors: E
authors: Rosini
authors: Loredano
authors: L
authors: Pollegioni
authors: Alice
authors: A
authors: Vrielink
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002511:Cephalosporins / Q000378:metabolism; D018360:Crystallography, X-Ray; D007700:Kinetics; D009154:Mutation; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002511:Cephalosporins; D011994:Recombinant Proteins; C025163:cephalosporin C; D012694:Serine; C030735:7-aminocephalosporanic acid; D000581:Amidohydrolases; D010399:Penicillin Amidase; C019672:aminoacylase I