Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit

• Published in: The Journal of biological chemistry 2002-11, Vol.277 (44), p.42121-42127
• Main Authors: Otten, Linda G, Sio, Charles F, Vrielink, Johanna, Cool, Robbert H, Quax, Wim J
• Format: Article
• Language: English
• Subjects: Mutagenesis; Penicillin Amidase - chemistry; Penicillin Amidase - genetics; Polymerase Chain Reaction; Protein Subunits; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M208317200

Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the beta-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.