An investigation of a quorum-quenching lactonase from Bacillus thuringiensis

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An investigation of a quorum-quenching lactonase from Bacillus thuringiensis

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dc.contributor.advisor Fast, Walter L.
dc.creator Momb, Jessica E.
dc.date.accessioned 2011-02-11T21:24:12Z
dc.date.accessioned 2011-02-11T21:24:34Z
dc.date.available 2011-02-11T21:24:12Z
dc.date.available 2011-02-11T21:24:34Z
dc.date.created 2009-12
dc.date.issued 2011-02-11
dc.date.submitted December 2009
dc.identifier.uri http://hdl.handle.net/2152/ETD-UT-2009-12-421
dc.description.abstract Gram-negative bacteria use N-acyl homoserine lactones (AHLs) to sense population density and regulate gene expression, including virulent phenotypes. The quorum-quenching AHL lactonase from Bacillus thuringiensis cleaves the lactone ring of AHLs, disabling this mode of gene regulation. Despite the potential applications of this enzyme as an antibacterial weapon, little was known about it's lactone ring-opening mechanism. As a member of the metallo-beta-lactamase superfamily, AHL lactonase requires two divalent metal ions for catalysis. NMR experiments confirm that these metal ions are also involved in proper enzyme folding. The chemical mechanism of ring opening was explored using isotope incorporation studies, and hydrolysis was determined to proceed via a nucleophilic attack by a solvent-derived hydroxide at the carbonyl of the lactone ring. A transient, kinetically significant metal-leaving group interaction was detected in steady-state kinetic assays with AHL lactonase containing alternative divalent metal ions hydrolyzing a sulfur-containing substrate. High-resolution crystal structures implicated two residues in substrate binding and hydrolysis, Tyr194 and Asp108. Site-directed mutagenesis of these residues followed by steady-state kinetic studies with wild-type and mutant enzymes hydrolyzing a spectrum of AHL substrates revealed that mutations Y194F and D108N significantly affect catalysis. Combining these results allows the proposal of a detailed hydrolytic mechanism. The binding site for the N-acyl hydrophobic moiety was probed using steady-state kinetics with a variety of naturally occurring and non-natural AHL substrates, and these studies indicate that AHL lactonase will accept a broad range of homoserine lactone containing substrates. Crystal structures with AHL substrates and non-hydrolyzable analogs reveal two distinct binding sites for this N-acyl group. Based on the ability of this enzyme to accommodate a variety of substrates, AHL lactonase was shown to have the ability to quench quorum sensing regulated by a newly discovered class of homoserine lactone signal molecules possessing an N-aryl group using a bioassay. Steady-state kinetic studies confirm that this class of signal molecules are indeed substrates for AHL lactonase.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subject Quorum-quenching
dc.subject Lactonase
dc.subject Metalloenyzme
dc.subject N-acyl homoserine lactone
dc.title An investigation of a quorum-quenching lactonase from Bacillus thuringiensis
dc.date.updated 2011-02-11T21:24:35Z
dc.contributor.committeeMember Hoffman, David
dc.contributor.committeeMember Johnson, Kenneth
dc.contributor.committeeMember Whitman, Christian
dc.contributor.committeeMember Zhang, Yan Jessie
dc.description.department Chemistry and Biochemistry
dc.type.genre thesis
dc.type.material text
thesis.degree.department Chemistry and Biochemistry
thesis.degree.discipline Biochemistry
thesis.degree.grantor The University of Texas at Austin
thesis.degree.level Doctoral
thesis.degree.name Doctor of Philosophy

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