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A Phenylalanine Clamp Controls Substrate Specificity in the Quorum-Quenching Metallo-γ-lactonase from Bacillus thuringiensis

Biochemistry2013Vol. 52(9), pp. 1603–1610

Citations Over TimeTop 11% of 2013 papers

Ce Feng Liu, Dali Liu, Jessica Momb, Pei W. Thomas, Ashley Lajoie, Gregory A. Petsko, Walter Fast, Dagmar Ringe

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.

Citations Over TimeTop 11% of 2013 papers

Abstract

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