Analysis of a substrate specificity switch residue of cephalosporin acylase.
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.
Sio
Charles F
CF
Pharmaceutical Biology, University Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Otten
Linda G
LG
Cool
Robbert H
RH
Quax
Wim J
WJ
D000272:Adipates / Q000737:chemistry; D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000737:chemistry*; D015226:Dinucleoside Phosphates / Q000737:chemistry*; D004789:Enzyme Activation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000272:Adipates; D002511:Cephalosporins; D015226:Dinucleoside Phosphates; D011994:Recombinant Proteins; C015101:cytidylyl-(3'-5')-cytidine; C081063:glutaryl-7-aminocephalosporanic acid; C014320:7-aminodesacetoxycephalosporanic acid; D010399:Penicillin Amidase