Burkholderia cenocepacia causes Cepacia syndrome, a fatal pneumonia that affects Cystic Fibrosis patients. Aromatic degradation has been linked to virulence in B. cenocepacia by insertional mutagenesis of genes involved in phenylacetic acid catabolism. B. cenocepacia has two paralogous copies of PaaK, the phenylacetyl-CoA ligase, which produces PA-CoA, the inducer of the pathway. Our objective was to assess a role for PaaK1 and PaaK2 in PA metabolism and virulence by constructing clean deletion mutants for each gene, and a double paaK mutant, as well as to quantify virulence using the nematode host model. Deletion and complementation of paaK1 revealed no change in killing phenotype. Reporter activity assays revealed PaaK1-dependent induction of the PA pathway 3-hydroxyphenylacetic acid but not 4-hydroxyphenylacetic acid. Altogether, these results demonstrate that 3-OHPA induces the PA degradation pathway in a paaK1 dependent manner and that PaaK1 is not involved in pathogenicity.

Burkholderia cenocepacia causes Cepacia syndrome, a fatal pneumonia that affects Cystic Fibrosis patients. Aromatic degradation has been linked to virulence in B. cenocepacia by insertional mutagenesis of genes involved in phenylacetic acid catabolism. B. cenocepacia has two paralogous copies of PaaK, the phenylacetyl-CoA ligase, which produces PA-CoA, the inducer of the pathway. Our objective was to assess a role for PaaK1 and PaaK2 in PA metabolism and virulence by constructing clean deletion mutants for each gene, and a double paaK mutant, as well as to quantify virulence using the nematode host model. Deletion and complementation of paaK1 revealed no change in killing phenotype. Reporter activity assays revealed PaaK1-dependent induction of the PA pathway 3-hydroxyphenylacetic acid but not 4-hydroxyphenylacetic acid. Altogether, these results demonstrate that 3-OHPA induces the PA degradation pathway in a paaK1 dependent manner and that PaaK1 is not involved in pathogenicity.

Thesis (Master of Applied Science in Chemistry)--Cape Peninsula University of Technology, 2018. / The prediction of the single crystal structure that will form due to the combination of two or more compounds to form a multicomponent crystal is one of the important areas of research in crystal engineering. Since these compounds display different properties when combined as a single crystal, knowledge of synthesis and design of the resulting compound is essential. The formation of a multicomponent crystal, such as a salt or a cocrystal generally depends on the complementarity of the functional groups present on both components. This means that basicity and acidity of the functional groups present on the selected compounds need to be considered. This study investigated salts and cocrystals of 3-chloro-4-hydroxyphenylacetic acid (CHPAA) using the ΔpKa rule. The calculated ΔpKa values were recorded and correlated with the experimental analysis in predicting the outcome of the crystallisation experiments ie. salt or cocrystal formation. This was further confirmed by the analysis of the C-O bond lengths found in the crystal structures. Salts were obtained by combinations of CHPAA with several organic bases (co-formers) such as diethylamine, dibutylamine, 2-aminopyridine, 2-amino-4-methylpyridine, 2-amino-6-methylpyridine and 4-dimethylaminopyridine. The calculated ΔpKa values were within the range of salt formation. Furthermore, the experimental analysis also showed that all resulting compounds were salts. Cocrystals were obtained by reactions of nicotinamide, isonicotinamide, phenazine and 4,4’-bipyridine with CHPAA. Again, the calculated ΔpKa values predicted cocrystals as the new solid forms. Experimental analysis carried out also confirmed cocrystal formation. For all resulting compounds, the comparison of intermolecular interactions as well as supramolecular synthons were reported. All compounds were synthesised by slow evaporation techniques using various organic solvents and characterised by single crystal X-ray diffraction, powder X-ray diffraction, thermal analysis and Fourier transformer infrared spectroscopy. From the structural analysis, it was found that all resulting structures displayed strong N-H•••O and O-H•••O intermolecular interactions including weak interactions of C-H•••Cl, C-H•••O and C-H•••π for a few of the structures. Furthermore, comparison of the crystal structures showed that no packing arrangement similarity existed between the compounds.

Aromatic compounds comprise approximately 25% of the Earth`s biomass. Accordingly, turnover of these thermostable compounds is essential to the biogeochemical carbon cycle. Specialized microbial enzymatic pathways are largely responsible for mineralization of aromatic compounds, and are extensively studied for bioremediation purposes. The phenylacetic acid degradation pathway (PAA) is of particular interest as it is utilized for degradation of a multitude of aromatic compounds, including environmental pollutants, and appears to be widely distributed among bacteria. Intriguingly, the PAA pathway has also been implicated as a virulence factor in the cystic fibrosis pathogen, Burkholderia cenocepacia. As such, detailed biochemical characterization of the PAA pathway holds great potential for improving bioremediation strategies for aromatic pollutants, as well as understanding carbon source utilization during B. cenocepacia infection. A striking feature of the PAA pathway in B. cenocepacia is the presence of two genes encoding the phenylacetate CoA ligase (PCL) enzyme (paaK1 and paaK2), responsible for the initial CoA activation of phenylacetic acid. PCLs are members of the adenylate forming enzyme superfamily. However, sequence alignments reveal several intriguing features, including a potentially novel microdomain consisting of the initial ~80 N-terminal residues, which possesses percent identity to any structurally characterized family member. Furthermore, this superfamily utilizes a complicated catalytic mechanism, exploiting several conserved motifs during the reaction process. The precise roles of many key conserved residues are not yet well understood, especially during the pre-adenylation, ATP bound state, for which few high quality crystal structures exist. In order to define the early stages of the catalytic mechanism, and to assess how the divergent polypeptide region may impact the PaaK enzymes, we have pursued a detailed structural characterization of the paralogs, complemented with functional assessments. Specifically, we have produced a 1.6 Å resolution crystal structure of PaaK1 in complex with ATP, which reveals a novel helical bundle arrangement at the N-terminal domain never before seen in this superfamily. Remarkably, homodimerization of PaaK1 appears to reconstitute potentially important β sheet interactions observed in the classical N-terminal arrangement of family members. Moreover, our structure is one of few which contain well ordered β and γ phosphates, allowing for detailed examination of significant protein-ATP interactions with conserved catalytic residues. To better comprehend the roles of these residues over the course of the reaction, we have produced additional crystal structures of PaaK1 and PaaK2 in complex with the phenylacetyl adenylate intermediate. Notably, PaaK2 was captured following the domain reorientation, poised to catalyze thioesterification of phenylacetyl adenylate, providing insight into the later stages of the reaction process. Furthermore, the intermediate co-structures divulge the location of the aryl substrate binding pocket for both paralogs. Detailed comparisons of the binding pockets accompanied kinetic characterizations for both paralogs, demonstrating that PaaK2 possesses an apparent KM of 150 μM for phenylacetic acid, more than double that of PaaK1 (62 μM). Our findings provide preliminary evidence for distinct functional roles of the PaaK paralogs in B. cenocepacia, while imparting additional insight into catalytic roles of conserved residues within the adenylate forming superfamily at large. / Graduate

The phenylacetic acid degradation pathway of Burkholderia cenocepacia is necessary for full pathogenicity of B. cenocepacia in nematode; however, the reasons of such requirements are unknown. Unlike wild type B. cenocepacia, a deletion mutant of the phenylacetyl-CoA monooxygenase complex (ΔpaaABCDE) released phenylacetic acid extracellularly in conditions that allow infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the ΔpaaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild type, due to phenylacetic acid consumption. Detection of acyl-homoserine lactones was reduced in spent medium from ΔpaaABCDE compared to that of the wild type strain. Phenotypes repressed in ΔpaaABCDE, protease activity and pathogenicity against C. elegans, increased with the addition of exogenous N-octanoyl-L-homoserine lactone. Thus, it was demonstrated that the attenuated phenotype of B. cenocepacia ΔpaaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N-octanoyl-L-homoserine lactone signaling. / October 2014

Thesis (Ph. D.)--University of Chicago. / "Reprinted from the Journal of the American Chemical Society, Vol. XLIII. No. 1. January 1921." Includes Abstract. Includes bibliographical references. Also available on the Internet.

This thesis deals with the preparation of dialkylaminoalkoxy derivatives of p-chlorophenoxyacetic acid and phenylacetic acid.

This thesis describes the creation of substituted diphenylacetic acids and their results.

No description available.

Mikroorganismen sind in der Lage, zahlreiche Xenobiotika abzubauen. Dazu nutzen sie unter aeroben Bedingungen oft einleitend Oxygenasen. Durch diese kann molekularer Luftsauerstoff aktiviert und auf organische Moleküle übertragen werden. Danach können die Verbindungen in den Metabolismus der Mikroorganismen eingeschleust und teils oder vollständig abgebaut werden. Am Beispiel des Styrols zeigen wir hier eine solche Abbauroute und wie wir diese biotechnologisch nutzen können, um interessante Verbindungen zu synthetisieren. Zielmoleküle der gesamten Enzymkaskade sind dabei diverse Phenylessigsäurederivate.