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Pyridine ring analogs of the aromatic amino acids phenylalanine and tyrosine were synthesized and studied in microbiological and mammalian systems.

The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.

The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.

Four casein-derived peptides fractions of varying hydrophobicity were obtained from á-chymotryptic digest of acid casein using hydrophobic interaction chromatography, termed fractions one through four (abbreviated, F1, F2, F3, and F4). Four standard methods involving alkoxyl, peroxyl, 2, 2-diphenyl-1-picrylhydrazl (DPPH), and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) ABTS•+ radicals, were used to measure antioxidative properties. While significantly superior efficacy was exhibited by F2 for all tests except against DPPH, no correlation between antioxidant efficacy and surface hydrophobicity was found. By using capillary electrophoresis and high performance liquid chromatography, the detection of aromatic chromophores by ultraviolet at 280 nm in the fractions revealed that F2 contained the highest concentration of aromatic amino acids and a unique peptide. Result from circular dichroism exhibited remaining residual structure in F2 compared with undigested casein. The F2 possesses a high potential to be used in food industry as a natural source of antioxidant with pronounced antioxidant capacity.

<p>Phenylalanine (Phe) is a proteinogenic aromatic amino acid that also serves as a precursor for numerous primary and secondary metabolites in plants. Phe is synthesized from chorismate, the final product of the shikimate pathway. In plants, Phe is predominantly synthesized in the plastids via the arogenate pathway, while most Phe-derived compounds are produced in the cytoplasm, requiring exportation of Phe from plastids to the cytosol. Here, we provided genetic evidences that a<i> Petunia hybrida</i> plastidial cationic amino acid transporter (PhpCAT) participates in the exportation of Phe from plastids, as well as regulation of carbon flux through Phe biosynthesis.</p> <p> By using reverse genetics, we demonstrated that a petunia phenylpyruvate aminotransferase (PhPPY-AT) is able to convert phenylpyruvate to Phe in the cytosol <i>in vivo</i>, and that a cytosolic chorismate mutase (CM2), which converts chorismate to prephenate, directs carbon flux from the plastidial Phe biosynthesis pathway towards the cytosolic pathway. Downregulation of <i>PhPPY-AT</i> and <i>PhCM2</i> resulted in significant decreases in Phe levels and emission of Phe-derived volatiles in petunia flowers, respectively. Metabolic flux analysis showed that the carbon flux through the cytosolic Phe biosynthesis pathway is significantly lower in <i>PhCM2</i> RNAi petunia flowers relative to wild type control. We also demonstrated that the conversion of prephenate to phenylpyruvate in the cytosol is catalyzed by a cytosolic prephenate dehydratase (PDT) produced from an alternative transcription start site of a known plastidial arogenate dehydratase (ADT). These results suggest that a microbial-like phenylpyruvate pathway for Phe biosynthesis operates in the cytosol of plant cells and the cytosolic pathway splits from the plastidial pathway at chorismate.</p> <p> To evaluate the metabolic potential of the cytosolic phenylpyruvate pathway, <i>PhCM2 </i>overexpressing transgenic petunia plants were generated. Unexpectedly, Phe levels and emission of Phe-derived volatiles were both reduced, even though the flux through the cytosolic pathway was increased relative to wild type control. Electron microscopy, metabolic profiling and metabolic flux analysis revealed that the number of leucoplasts, starch levels and flux through the plastidial pathway were all reduced in <i>PhCM2</i> overexpression lines, while the concentrations of auxin and its biosynthetic intermediate, indole-3-pyruvic acid (IPA), were elevated. Overexpression of Arabidopsis aminotransferase VAS1, which converts IPA to Trp, in <i>PhCM2</i> overexpression petunia background recovered Phe levels and Phe-derived volatiles emission. These results indicate that there exists a metabolic crosstalk between cytosolic Phe production and Trp-dependent auxin biosynthesis .</p> <p> Our research completed the post-chorismate cytosolic Phe biosynthesis pathway in plants and revealed possible metabolic crosstalk between cytosolic Phe production and auxin biosynthesis in plant cells, providing targets for future genetic modification of metabolites in plants.</p>

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a primary constituent of chronic, polymicrobial infections in the lungs of individuals with cystic fibrosis (CF). A significant consequence of CF is production of thick mucus along epithelial surfaces. In the lungs, this mucus collects and serves as an excellent growth substrate for a range of bacteria including. CF lung fluids (sputum) also enhance the virulence of P. aeruginosa, as production of a signaling molecule critical for virulence, the Pseudomonas quinolone signal (PQS), is enhanced in the presence of phenylalanine and tyrosine in CF sputum. The goal of this dissertation is to better understand how phenylalanine and tyrosine affect PQS production and ultimately P. aeruginosa virulence. To address this, I use transcriptome profiling to determine that genes for phenylalanine and tyrosine catabolism, PQS biosynthesis, and a transcriptional regulator called PhhR are up-regulated in the presence of phenylalanine and tyrosine. I determine that PhhR regulates genes for aromatic amino acid catabolism but not genes for PQS biosynthesis. The PhhR regulon is further characterized by mapping of PhhR-regulated promoters with primer extension, and evidence for direct regulation is presented. To explain enhanced production of PQS in CF sputum, I favor a model in which flux of a shared metabolic precursor, chorismate, toward PQS biosynthesis is enhanced when phenylalanine and tyrosine are present. I investigate this model by examining the first step in PQS biosynthesis, conversion of chorismate to anthranilate by an anthranilate synthase (AS). P. aeruginosa possesses two AS enzymes encoded by the trpEG and phnAB genes, with the former generating anthranilate specifically for tryptophan biosynthesis while the latter generates anthranilate for PQS biosynthesis. I investigate the evolutionary origins of these two enzymes and generate unmarked deletion mutants to dissect their roles in tryptophan and PQS biosynthesis. The ability of PhnAB to compensate for loss of TrpEG at high cell densities is documented, and a model explaining anthranilate sequestering is developed. Knowledge gained from these studies will be useful in developing novel therapeutic strategies. / text

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The shikimate pathway is a biosynthetic pathway that is responsible for producing a variety of organic compounds that are necessary for life in plants and microorganisms. The pathway consists of seven enzyme catalysed reactions beginning with the condensation reaction between D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to give the seven-carbon sugar DAH7P. This thesis describes the design, synthesis and evaluation of a range of alternative non-natural four-carbon analogues of E4P (2- and 3-deoxyE4P, 3-methylE4P, phosphonate analogues of E4P) to probe the substrate specificity of different types of DAH7P synthases [such as Mycobacterium tuberculosis (a type II DAH7PS), Escherichia coli (a type Ialpha DAH7PS) and Pyrococcus furiosus (a type Ibeta DAH7PS)].