Browsing by Subject "Quorum-quenching"

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Investigation and engineering of PvdQ, a Pseudomonas aeruginosa enzyme at the nexus of quorum sensing and iron uptake pathways
(2014-12) Clevenger, Kenneth David; Fast, Walter L.; Whitman, Christian P; Whiteley, Marvin; Keatinge-Clay, Adrian; Zhang, Jessie Y
The gram-negative human pathogen Pseudomonas aeruginosa is a widespread global health concern. Two key pathways of P. aeruginosa that are involved in its infection and/or survival within mammalian hosts are the pyoverdine biosynthetic pathway, which is necessary for iron acquisition, and the N-acyl-homoserine lactone (AHL) quorum sensing (QS) system, which is necessary for P. aeruginosa virulence and infection. The P. aeruginosa enzyme PvdQ is unique in that it is proposed to play roles in both these pathways. To investigate the role of PvdQ in P. aeruginosa, the enzyme’s in vitro substrate profile was characterized and found to favor substrates with unsubstituted myristic acyl groups, supporting a role for PvdQ in pyoverdine biosynthesis, but challenging its proposed role in regulating P. aeruginosa QS. Knowledge of PvdQ’s substrate preference and mechanism enabled rational inhibitor design, leading to the discovery of long chain (≥ 10 carbons) n-alkylboronic acids as highly potent transition state analog inhibitors of PvdQ, with 1-tridecylboronic acid having a Ki of 200 ± 40 pM and having the ability to recapitulate the growth phenotype of a pvdQ transposon insertion strain of P. aeruginosa under certain experimental conditions. Investigation of PvdQ inhibition by alkylboronic acids was extended to short- and medium-chain alkylboronic acids and revealed an eight order of magnitude span of affinity that is linearly related to alkyl chain length. Through structural studies, short-chain alkylboronic acids (≤ 4 carbons) were found to adopt an alternate binding conformation relative to other alkylboronic acids, which suggests a mechanism by which PvdQ excludes short substrates such as P. aeruginosa QS molecule N-butyroyl-homoserine lactone. Finally, a circular permutation of PvdQ (cpPvdQ) was designed and constructed in order to uncouple the catalytic and self-processing activities of PvdQ and to facilitate the production of mutants that affect catalysis and specificity. The cpPvdQ monomer recapitulates the catalytic and structural features of PvdQ. A catalytically impaired cpPvdQ mutant that is shown to maintain affinity for PvdQ’s native substrate, the pyoverdine precursor, but does not hydrolyze the substrate, has been produced and will be a valuable tool for future efforts to trap a PvdQ derivative in complex with the pyoverdine precursor.
An investigation of a quorum-quenching lactonase from Bacillus thuringiensis
(2009-12) Momb, Jessica E.; Fast, Walter L.; Hoffman, David; Johnson, Kenneth; Whitman, Christian; Zhang, Yan Jessie
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.