Analysis of a substrate specificity switch residue of cephalosporin acylase |
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Authors: | Sio Charles F Otten Linda G Cool Robbert H Quax Wim J |
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Institution: | Pharmaceutical Biology, University Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. |
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Abstract: | Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( Formula: see text] (cat)/ Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of Formula: see text] (m). The Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain. |
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Keywords: | Cephalosporin acylase Glutaryl acylase Substrate specificity Protein engineering Site directed mutagenesis Saturation mutagenesis Adipyl-7-ADCA Semi-synthetic cephalosporins Pseudomonas SY-77 |
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