An investigation of a quorum-quenching lactonase from Bacillus thuringiensis

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Title: An investigation of a quorum-quenching lactonase from Bacillus thuringiensis
Author: Momb, Jessica E.
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 .
URI: http : / /hdl .handle .net /2152 /ETD -UT -2009 -12 -421
Date: 2011-02-11

Citation

An investigation of a quorum-quenching lactonase from Bacillus thuringiensis. Doctoral dissertation, The University of Texas at Austin. Available electronically from http : / /hdl .handle .net /2152 /ETD -UT -2009 -12 -421 .

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