Altering the Substrate Specificity of Cephalosporin Acylase by Directed Evolution of the β-Subunit

Abstract

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 β-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-ADCAversus 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-77N266H 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-77F375L 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. 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 β-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-ADCAversus 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-77N266H 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-77F375L 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. 7-aminocephalosporanic acid 7-aminodesacetoxycephalosporanic acid adipyl-7-ADCA glutaryl-7-ACA error-prone PCR wild type minimal agar supplemented with adipyl-leucine minimal agar supplemented with glutaryl-leucine minimal agar supplemented withl-leucine minimal agar without leucine source hydrolysis rate of AD-7-ADCA/hydrolysis rate of GL-7-ACA Semisynthetic cephalosporins and penicillins are the most widely used antibiotics. All clinically important semisynthetic cephalosporins are made from 7-aminocephalosporanic acid (7-ACA)1 or 7-aminodesacetoxycephalosporanic acid (7-ADCA). 7-ACA is derived from cephalosporin C (aminoadipyl-7-ACA), which is obtained by fermentation of the fungus Cephalosporium acremonium. Deacylation is performed either chemically or by a two-step enzymatic process using ad-amino acid oxidase and a glutaryl acylase. The latter enzyme can be found in several Pseudomonas andAcinetobacter species (1Aramori I. Fukagawa M. Tsumura M. Iwami M. Isogai T. Ono H. Ishitani Y. Kojo H. Kohsaka M. Ueda Y. Imanaka H. J. Ferment. Bioeng. 1991; 72