Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.D.)--University of Adelaide, Faculty of Medicine, 1984. / Articles and serials related to the thesis research bound in appendix. Includes bibliographical references.

The catalytic and regulatory mechanisms of phenylalanine hydroxylase were investigated by structural studies of in this research. Phenylalanine hydroxylase (PheH) hydroxylates phenylalanine to produce tyrosine using tetrahydrobiopterin (BH4) and oxygen. The three ligands to the iron, His285, His290, and Glu330, were mutated to glutamine, glutamate, and histidine. All the mutants had low but measurable activity. Mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. All the mutants greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family. Hydrogen/deuterium exchange and mass spectrometry showed that peptides lying in the interface between the regulatory and catalytic domains display large increases of deuterium incorporation in the presence of phenylalanine. However, the effects of phenylalanine on a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site of phenylalanine. These results support the autoinhibitory function of the N-terminus of PheH. No peptides show a changed deuterium incorporation pattern in the presence of BH4, suggesting that BH4 binding does not change the structure significantly from the resting form. In phosphorylated PheH, three peptides show a deuterium incorporation pattern similar to that of unphosphorylated PheH plus phenylalanine, while the other peptides sensitive to phenylalanine binding in unphosphorylated PheH show the same pattern as that of unphosphorylated PheH without phenylalanine. Therefore, the conformational changes induced by phosphorylation are similar to but not identical to those associated with phenylalanine activation. The isolated regulatory domain (PheH1-117) was expressed and purified using a QSepharose column followed by a gel filtration column. Analytical gel filtration shows that PheH1-117 exists as a dimer in solution. In the presence of phenylalanine, the retention time of PheH1-117 is significantly changed. The 1H-15N NMR spectra of PheH1- 117 show that the cross-peaks of several residues are altered in the presence of phenylalanine. These results support the existence of a regulatory binding site for phenylalanine in the regulatory domain of PheH.

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

No description available.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Thesis--Catholic University of America. / Bibliography: p. 45-51.

Induction of phenylpropanoid metabolism in many plants is associated with the induction of plant defence responses. Among these are the accumulation of phenylpropanoid-derived phytoalexins, increase in lignification around infected sites, and the accumulation of wall-bound phenolic compounds. I show in this work, that H11 hybrid cell suspension cultures when treated with either of three elicitors respond with an increase in phenylpropanoid metabolism. Activation proceeds rapidly from PAL and 4CL mRNA accumulation, to a massive increase in extractable PAL enzyme activity and finally there is accumulation of specific phenolic compounds in the cell extracts, culture filtrates, and cell walls. In addition, elicitor treatment causes cells to turn brown, indicative of phenolic compound accumulation. As in other plants, induction is dependent on culture age, is dose dependent, and the kinetics of induction is the same with all three elicitors. Based on the previously established mode of action of PGA lyase as an elicitor, it is concluded that in poplar, as in other plants, defence responses can be induced by elicitors from both fungal and plant cell wall origin. These results illustrate the successful use of plant suspension cultures as a simplified system to study inducible defence responses. In addition, and consistent with the ubiquitous nature of phenolics in poplar, phenylpropanoid metabolism may play an important role in plant defence responses in this species. / Science, Faculty of / Botany, Department of / Graduate

The enzymes phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were studied in the yeast Sporobolomyces roseus (Kluyver and van Neil). Cells grown on a glucose-salts medium were ground with alumina, and the cell-free buffer extract was fractionated with ammonium sulfate. Enzyme activity was assayed by measuring spectrophotometrically the cinnamic acid and p-coumaric acid produced from phenylalanine and tyrosine respectively. Further attempts at purification resulted in the inactivation of the TAL. Although the two enzymes were not separated by the purification procedures used, there is some evidence that the deamination of phenylalanine and tyrosine are catalyzed by different proteins, and not by a single enzyme with wide specificity. TAL appears to be precipitated by lower concentrations of ammonium sulfate than is PAL. The pH curves of the two enzymes are different. The specific activities of the two enzymes can be changed relative to one another in the cell by changing the medium upon which the cells were grown. The rates of production of the two enzymes vary independently during the growth of the cells. While the proteins are probably distinct, the production and activity of each enzyme seem to be under common control. Peak production of both enzymes occurs during late logarithmic-early stationary phase in the growth of a batch culture. Replacement media containing either phenylalanine or tyrosine stimulate the production of both PAL and TAL. Similarly, media containing cinnamic acid or p-coumaric acid repress the formation of the enzymes. Studies using labelled substrate show that both products inhibit the action of both enzymes. / Science, Faculty of / Botany, Department of / Graduate