Show simple item record

dc.creatorMomb, Jessica E.
dc.date.accessioned2011-02-11T21:24:12Z
dc.date.accessioned2011-02-11T21:24:34Z
dc.date.available2011-02-11T21:24:12Z
dc.date.available2011-02-11T21:24:34Z
dc.date.created2009-12*
dc.date.issued2011-02-11
dc.date.submittedDecember 2009
dc.identifier.urihttp://hdl.handle.net/2152/ETD-UT-2009-12-421
dc.descriptiontext
dc.description.abstractGram-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.mimetypeapplication/pdf
dc.language.isoeng
dc.subjectQuorum-quenching
dc.subjectLactonase
dc.subjectMetalloenyzme
dc.subjectN-acyl homoserine lactone
dc.titleAn investigation of a quorum-quenching lactonase from Bacillus thuringiensis
dc.date.updated2011-02-11T21:24:35Z
dc.description.departmentChemistry and Biochemistry
dc.type.genrethesis*
thesis.degree.departmentChemistry and Biochemistry
thesis.degree.disciplineBiochemistry
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy


Files in this item

Icon

This item appears in the following Collection(s)

Show simple item record