A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

• Published in: Biotechnology letters 2021-07, Vol.43 (7), p.1467-1473
• Main Authors: Murugayah, Shereen A., Evans, Gary B., Tyndall, Joel D. A., Gerth, Monica L.
• Format: Article
• Language: English
• Subjects: Amino acids; Aminocephalosporanic acid; Applied Microbiology; Biochemistry; Biomedical and Life Sciences; Biotechnology; Crystal structure; Glycine; Homoserine lactones; Lactones; Life Sciences; Microbiology; Mutagenesis; Mutation; N-Acyl homoserine lactone; Original Research Paper; Point mutation; Quorum sensing; Residues; Saturation mutagenesis; Site-directed mutagenesis; Substrate specificity; Substrates
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-021-03135-9

Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N -acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N -acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N -acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.