title: Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate modeling and active site mutations.
abstract: The N-acyl- l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-beta-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme-product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.
authors.affiliation: Graduate Program in Biochemistry, The University of Texas, Austin, Texas 78712, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Momb
authors.forename: Canhui
authors.initials: C
authors.lastname: Wang
authors.forename: Dali
authors.initials: D
authors.lastname: Liu
authors.forename: Pei W
authors.initials: PW
authors.lastname: Thomas
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Petsko
authors.forename: Hua
authors.initials: H
authors.lastname: Guo
authors.forename: Dagmar
authors.initials: D
authors.lastname: Ringe
authors.forename: Walter
authors.initials: W
authors.lastname: Fast
mesh_terms: D001413:Bacillus thuringiensis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D053038:Quorum Sensing
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; C432003:N-acyl homoserine lactonase