Autoinducer inactivator A (AiiA) is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity but shows a preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and the F107W mutation results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrate’s N-acyl chain. Two aromatic residues, F64 and F68, form a hydrophobic clamp, centered around the seventh carbon in the product-bound structure’s decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox-sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics.

A tetrafluorophenol acrylamide monomer unit was synthesised, co-polymerised and grafted onto a glass slide to form individual gel spots. As a proof of principle study, a small library of amides was rapidly synthesised within these gel spots using ‘catch-and-release’ chemistry, including the biologically interesting quorum sensing acyl-homoserine lactones. The tetrafluorophenol acrylamide gel provides an efficient platform to synthesise and screen small molecules for biological activity.The development of tetrafluorophenol acrylamide 3D gels as an effective platform for the synthesis of small molecules has been achieved. Furthermore, this offers the potential to synthesise and screen compounds for biological activity on the same slide.