Journal
BIOTECHNOLOGY LETTERS
Volume 43, Issue 7, Pages 1467-1473Publisher
SPRINGER
DOI: 10.1007/s10529-021-03135-9
Keywords
Glutaryl-7-aminocephalosporanic acid acylase; N-acyl-homoserine lactone acylase; Protein engineering; Quorum quenching; Site‐ saturation mutagenesis
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Funding
- University of Otago Doctoral Scholarship
- New Zealand Ministry of Business Innovation and Employment Smart Ideas Grant [RTVU1504]
- New Zealand Ministry of Business, Innovation & Employment (MBIE) [RTVU1504] Funding Source: New Zealand Ministry of Business, Innovation & Employment (MBIE)
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By site-directed mutagenesis, the substrate specificity of a glutaryl-7-aminocephalosporanic acid acylase was changed from having no activity against N-acyl homoserine lactones to having new activities. This approach may be useful for further engineering of 'quorum quenching' enzymes.
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.
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