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

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.

Engineering quorum quenching enzymes: progress and perspectives.

Quorum sensing is a key contributor to the virulence of many important plant, animal and human pathogens. The disruption of this signalling-a process referred to as 'quorum quenching'-is a promising new approach for controlling microbial pathogens. In this mini-review, we have focused on efforts to engineer enzymes that disrupt quorum sensing by inactivating acyl-homoserine lactone signalling molecules. We review different approaches for protein engineering and provide examples of how these engineering approaches have been used to tailor the stability, specificity and activities of quorum quenching enzymes. Finally, we grapple with some of the issues around these approaches-including the disconnect between in vitro biochemistry and potential in vivo applications.

Optimisation of a high-throughput fluorescamine assay for detection of N-acyl-l-homoserine lactone acylase activity.

N-acyl-l-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-negative bacteria by degrading AHL signalling molecules. This degradation of signalling molecules (termed 'quorum quenching') has potential uses in the prevention or reduction of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterisation of quorum quenching enzymes. Here, we present an optimised fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format.