title: A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-γ-lactonase from Bacillus thuringiensis.
abstract: 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.
authors: Department of Chemistry and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.
authors: Ce Feng
authors: CF
authors: Liu
authors: Dali
authors: D
authors: Liu
authors: Jessica
authors: J
authors: Momb
authors: Pei W
authors: PW
authors: Thomas
authors: Ashley
authors: A
authors: Lajoie
authors: Gregory A
authors: GA
authors: Petsko
authors: Walter
authors: W
authors: Fast
authors: Dagmar
authors: D
authors: Ringe
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001413:Bacillus thuringiensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006714:Homoserine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D053038:Quorum Sensing; D013379:Substrate Specificity
chemical_list: D010649:Phenylalanine; D006714:Homoserine; D000581:Amidohydrolases; C515498:metallo-gamma-lactonase
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