Quorum sensing refers to bacterial systems in which a signal molecule is produced by a bacteria and diffuses into the environment. This allows for behavior to be controlled in a cell density dependent manner. Quorum sensing systems utilizing Acyl Homoserine Lactones (AHLs) are prominent in many microbial organisms and communities of high impact to human interests, such as agriculture, biofilm production, and certain pathogens. Interestingly, AHL chemical structures are variable and determine the specificity of the signal. Several strategies that interfere with quorum sensing are naturally used by various organisms. One such strategy is to use enzymes which degrade the AHL signal, known as Quorum Quenching (QQ). Previously characterized QQ enzymes have exhibited broad range AHL specificity. To target QQ approaches, we decided to create QQ enzymes with greater AHL specificity. This presents a challenge, as we wish to avoid altering the active site catalysis, while altering the specificity of the hydrophobic acyl chain of the substrate. We used the broad spectrum, thermostable lactonase from Parageobacillus caldoxylosilyticus, dubbed GcL, as a starting point of our rational design approach. Key residues along the substrate binding cleft which interact with the AHL substrate acyl chains were identified by structural studies and used as saturation library targets. Many mutants with altered substrate preference were identified in a screening step and confirmed with purified enzyme and kinetic characterization. Several mutations were combined and the best mutants obtained thus far have shifted substrate preference by up to 200-fold in favor of short chain AHLs. These mutants were crystallized and their structures revealed a reshaping of the active site binding cleft, as well as an alternate substrate binding conformation for a prominent active site loop.

Engineering of a quorum quenching lactonase for altered substrate specificity by rational design

ACS Spring 2022

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