A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis

• Published in: Biochemistry (Easton) 2013-02, Vol.52 (9), p.1603-1610
• Main Authors: Liu, Ce Feng, Liu, Dali, Momb, Jessica, Thomas, Pei W., Lajoie, Ashley, Petsko, Gregory A., Fast, Walter, Ringe, Dagmar
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi400050j

AiiA is a metal-dependent N -acyl homoserine lactone hydrolase that displays broad substrate specificity, but shows 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 F107W 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.