Mutational Analysis of a Key Residue in the Substrate Specificity of a Cephalosporin Acylase

Abstract

beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specificity. In order to explore the function of this residue in substrate specificity, we performed a complete mutational analysis of position 266. Codons for all amino acids were introduced in the gene, 16 proteins that could be functionally expressed in Escherichia coli were purified to homogeneity and their catalytic parameters were determined. The mutant enzymes displayed a broad spectrum of affinities and activities, pointing to the flexibility of the enzyme at this position. Mutants in which Asn266 was changed into Phe, Gln, Trp and Tyr displayed up to twofold better catalytic efficiency (k(cat)/K(m))than the wild-type enzyme when adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) was used as substrate, due to a decreased K(m). Only mutants SY-77(N266H) and SY-77(N266M) showed an improvement of both catalytic parameters, resulting in 10- and 15-times higher catalytic efficiency with adipyl-7-ADCA, respectively. Remarkably, the catalytic activity (k(cat)) of SY-77(N266M) when using adipyl-7-ADCA as substrate was as high as when glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was used, and approaches commercially interesting activity. SY-77(N266Q), SY-77(N266H) and SY-77(N266M) mutants showed a modest improvement in hydrolysing Cephalosporin C. Since these mutants also have a good catalytic efficiency when adipyl-7-ADCA is used and are still active towards glutaryl-7-ACA, they can be regarded as broad substrate acylases. These results demonstrate that the combination of directed evolution for the identification of important positions, together with saturation mutagenesis for finding the optimal amino acid, is a very effective method for finding improved biocatalysts.

Quorum Quenching by an N -Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

“…This strongly suggests that the PA2385 protein, like the ␤-lactam acylases, is an Nterminal nucleophile hydrolase, with the first residue of the ␤-subunit, serine, as the catalytically active residue (19,25). During purification, moreover, a protein that presumably is a partially maturated precursor in which the spacer peptide has not been cleaved from the ␣-subunit, as has been observed in mutants of cephalosporin acylase, was obtained (18,32). A variable cleavage site between the ␣-subunit and the spacer peptide, resulting in a heterogeneous enzyme sample, was observed.…”

Evidence for Niche Partitioning Revealed by the Distribution of Sulfur Oxidation Genes Collected from Areas of a Terrestrial Sulfidic Spring with Differing Geochemical Conditions

“…When mapped onto the SoxB enzyme structure of T. thermophilus, most of the amino acid sequence differences in the soxB genes from Rattlesnake Spring are found on the outer portion of the enzyme, generally away from the active site. Although changes in the outer structure of the enzyme might seem unlikely to induce a functional change in an enzyme, previous findings suggest that it is possible (60,61) and that even a single-amino-acid change can alter an enzyme's capabilities (62)(63)(64). For instance, the substitution of six amino acids (Ala, Asp, Cys, Lys, Phe, and Ser) was found to lead to improved environmental stability in the serine protease from Bacillus amyloliquefaciens that allowed the proteases to resist inactivation under higher temperatures and alkalinity (65).…”

Directed evolution: selecting today's biocatalysts

“…2) [19]. This directed evolution step guided us to the important residues, which we subsequently randomised into all 19 others to find the best amino acid at each position [20,21]. Determining the catalytic parameters from each purified enzyme on adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) revealed a single mutant having a 15 times improved catalytic efficiency resulting from an improved K m and a k cat similar to the k cat of wild type enzyme on glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA).…”