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, Ser1β, 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.
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Research Article|
March 28 2013
Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity
Emily Golden;
Emily Golden
*School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
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Rachel Paterson;
Rachel Paterson
*School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
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Wan Jun Tie;
Wan Jun Tie
*School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
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Anandhi Anandan;
Anandhi Anandan
*School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
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Gavin Flematti;
Gavin Flematti
*School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
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Gianluca Molla;
Gianluca Molla
†Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy
‡Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano, ICRM-CNR Milano and Università degli studi dell’Insubria, Italy
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Elena Rosini;
Elena Rosini
†Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy
‡Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano, ICRM-CNR Milano and Università degli studi dell’Insubria, Italy
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Loredano Pollegioni;
Loredano Pollegioni
†Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy
‡Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano, ICRM-CNR Milano and Università degli studi dell’Insubria, Italy
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Alice Vrielink
Alice Vrielink
1
*School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
1To whom correspondence should be addressed (email alice.vrielink@uwa.edu.au).
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Publisher: Portland Press Ltd
Received:
November 12 2012
Revision Received:
February 01 2013
Accepted:
February 04 2013
Accepted Manuscript online:
February 04 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 451 (2): 217–226.
Article history
Received:
November 12 2012
Revision Received:
February 01 2013
Accepted:
February 04 2013
Accepted Manuscript online:
February 04 2013
Citation
Emily Golden, Rachel Paterson, Wan Jun Tie, Anandhi Anandan, Gavin Flematti, Gianluca Molla, Elena Rosini, Loredano Pollegioni, Alice Vrielink; 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 15 April 2013; 451 (2): 217–226. doi: https://doi.org/10.1042/BJ20121715
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