Analysis of a substrate specificity switch residue of cephalosporin acylase

作者：Sio CF; Otten LG; Cool RH; Quax WJ^*

来源：Biochemical and Biophysical Research Communications, 2003, 312(3): 755-760.

DOI：10.1016/j.bbrc.2003.10.180

• cephalosporin acylase
• glutaryl acylase
• substrate specificity
• protein engineering
• site directed mutagenesis
• saturation mutagenesis
• adipyl-7-ADCA
• semi-synthetic cephalosporins
• Pseudomonas SY-77

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 (k(cat)/K-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 K-m. The k(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.

• 出版日期2003-12-19