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N-hydroxylating monooxygenases (NMO) are members the class B flavoprotein monooxygenases. They catalyze the N-hydroxylation of lysine and ornithine and play and essential role in the biosynthesis of hydroxamate containing siderophores. Siderophores are high affinity iron-chelators composed of catechol and hydroxamate functional groups that are synthesized and secreted by several microorganisms and plants. It has been showed that many NMOs are essential for virulence in many opportunistic pathogens such as Aspergillus fumigatus and Pseudomonas aeruginosa. The focus of my research is on the N-hydroxylating enzymes: Siderophore A (SidA) from Aspergillus fumigatus and Amycolatoposis alba monooxygenase (AMO). One of my projects is focusing on identifying inhibitors of SidA that will ultimately block the siderophore biosynthesis in A. fumigatus. Out of 973 compounds screened using an activity high-throughput assays two compounds were identified. These were, wortmannin a steroid metabolite and ebselen a benzoselenazole as SidA inhibitors with IC50 values of 369 µM and 11 µM respectively. A second part of this works investigates the hydroxamate formation of the siderophore albachelin in Amycolatoposis alba with the purpose of better understanding this class of enzymes and their catalytic mechanism. The enzyme was purified and characterized in its holo (FAD-bound) and apo (unbound) forms. Pre-steady and steady state kinetics shows that the two forms have different coenzyme preference; apo-AMO prefers NADH while holo-AMO has a higher affinity to NADPH. / Master of Science in Life Sciences / N-Hydroxylating monooxygenases (NMOs) are unique class of enzymes which are involved in the production of small iron binding molecules known as siderophore. Siderophores are used by some fungi and bacteria to acquire iron from the human host during infection. This thesis focuses on the inhibition and characterization of Siderophore A (SidA) from Aspergillus <i>fumigatus</i> and <i>Amycolatopsis alba</i> monooxygenases (AMO). SidA is an enzyme of interest because it is required for siderophore production and has been shown to be required for pathogenesis in <i>A. fumigatus</i>. In this thesis, a drug-discovery processes known as highthroughput screening was used to identify potential inhibitors of SidA. Using a several other assays, two compounds were validated as inhibitors of <i>A. fumigatus</i>. In addition, this work also investigates the reaction of AMO by focusing on the different stages of the enzyme mechanism; such as oxygen consumption, product formation, and flavin reduction. Overall, the goal of this thesis is to provide insight into the N-hydroxylating monooxygenases class of enzymes as well as to find new compounds that can lead to the next generation of antifungal drugs.

Plusieurs acteurs moléculaires impliqués dans les variations de qualité de la viande ont été récemment mis en évidence chez le poulet. Ma thèse a pour objectif d’approfondir l’étude de leur régulation en étudiant l’impact de facteurs alimentaires en interaction avec l’origine génétique des animaux. Il s’est articulé autour de deux thématiques qui impliquent des acteurs moléculaires et des critères de qualité de viande indépendants : le rôle de l’AMPK (AMP-activated protein kinase) dans le contrôle du turnover du glycogène musculaire et des caractères qui en dépendent (pH, rétention d’eau, couleur) et l’implication de BCMO1 (β, β-carotene-15,15’-monooxygenase) dans les variations de teneurs en pigments caroténoïdes et de coloration. Nos résultats soulignent dans les deux cas la possibilité de moduler les caractères de qualité via l’alimentation avec des réponses qui dépendent des caractéristiques génétiques des animaux. Nos travaux ont aussi permis d’améliorer la compréhension de la régulation des biomarqueurs étudiés par les nutriments et la génétique et contribueront à terme à la mise en place de nouvelles stratégies de production permettant d’optimiser la qualité du poulet de chair en réponse aux attentes de la filière et des consommateurs. / Several molecular mechanisms involved in the variations of poultry meat quality were recently identified in chickens. My thesis aims to further study their regulation by exploring the impact of dietary factors in interaction with the genetic origin of animals. It was structured around two themes that involve independent molecular mechanisms and meat quality criteria: the role of AMPK (AMP-activated protein kinase) in the control of muscle glycogen turnover and related meat traits (pH, water retention, color), and the involvement of BCMO1 (β, β-carotene-15, 15'-monooxygenase) in controlling levels of carotenoid pigments and yellow color. Our results emphasize in both cases the possibility of modulating quality traits through nutrition, with effects that depend on the genetic characteristics of animals. Our work has also improved the understanding of the regulation of studied biomarkers by genetics and nutrients. This should contribute to the development of new production strategies to optimize the quality of broilers in response to expectations of poultry producers and consumers.

O tetracloroeteno (PCE), um dos principais contaminantes de águas subterrâneas, é uma molécula recalcitrante, com toxicidade elevada. Processos de biorremediação de água ou solo contaminados com PCE são normalmente limitados pela baixa eficiência de microrganismos sabidamente envolvidos em sua degradação. No entanto, a prospecção de novos microrganismos, mais eficientes na degradação do PCE é uma alternativa para otimizar esses processos. Os objetivos deste estudo foram desenvolver uma técnica de biorremediação utilizando um reator horizontal de leito fixo (RHLF) contendo consórcios de microrganismos eficientes na degradação do PCE, e caracterizar a via de degradação do PCE utilizada pelo consórcio selecionado. Para tanto, amostras de sedimento de dois poços de monitoramento de água foram coletadas de uma metalúrgica com histórico de contaminação com PCE. Os sedimentos foram imobilizados, acondicionados em RHLFs específicos e submetidos a cultivo de enriquecimento em meio mínimo suplementado com PCE. A estrutura das comunidades e a diversidade bacteriana dos RHLFs foram avaliadas e comparadas com as amostras do PM1 e PM2, por PCR-DGGE e sequenciamento de bibliotecas de clones do gene rRNA 16S. Os resultados evidenciaram a seleção de populações bacterianas no RHLF contendo o inóculo do PM1 (In1) após o cultivo de enriquecimento, enquanto no RHLF contendo o inóculo do PM2 (In2), a estrutura da comunidade bacteriana não diferiu daquela observada no PM2. Ensaios de degradação do PCE nos RHLFs, usando cromatografia gasosa associada à espectrometria de massas (CG/EM), mostraram, após 12 horas, uma eficiência de 87 % na degradação do PCE no reator com In1 e 96 % no reator com In2. Foi feito o isolamento e identificação, por sequenciamento do gene rRNA 16S, das bactérias dos RHLFs, sendo identificados 4 isolados do In1, similares a Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans e Stenotrophomonas maltophilia e 7 isolados do In2, similares a Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii e Comamonas sp. Compostos orgânicos voláteis nos reatores com In1 e In2 foram analisados por CG/EM, identificando a produção de clorofórmio (TCM) e 1,1,1-tricloroetano (TCA) como produtos da degradação do PCE. Consórcios formados por bactérias isoladas dos reatores In1 (IIn1) e In2 (IIn2) foram imobilizados e acondicionados em RHLFs distintos para avaliar o potencial dos mesmos na degradação do PCE. Após 12 horas, 92 % do PCE foi degradado nos reatores com IIn1 e IIn2, com produção de TCM e TCA. Testes de degradação usando células em suspensão foram conduzidos para avaliar a eficiência de cada isolado na degradação do PCE. O isolado I8 do In2 (I8In2), identificado como Comamonas sp., teve 68 % de eficiência na degradação do PCE. Ensaios com inibidor de monoxigenases do citocromo P-450 (1-aminobenzotriazole) mostraram que a degradação do PCE nos RHLFs, contendo IIn1, IIn2 e I8In2, foram dependentes dessa enzima. Como conclusão, nós identificamos uma nova via de degradação do PCE altamente eficiente, aeróbia e mediada por monoxigenases e isolamos cepas bacterianas que podem ser usadas como consórcios imobilizados nos RHLFs como uma alternativa eficiente na remediação de áreas contaminadas com PCE. / Tetrachloroethene (PCE), one of the main contaminants of groundwater, is a recalcitrant molecule with high toxicity. Bioremediation processes of water or soil contaminated with PCE are usually limited by the low efficiency of microorganisms known to be involved in its degradation. However, the exploration of new and more efficient microorganisms in the degradation of PCE is an alternative to optimize these processes. The objectives of these studies were to develop a bioremediation technique using horizontal fixed bed reactor (HFBR) containing microbial consortia effective in the PCE degradation, and to characterize the PCE degradation pathway used by the selected consortium. For that, sediment samples of two groundwater monitoring wells were collected from a metallurgical plant with historical of PCE contamination. The sediments were immobilized, packed in specific HFBRs and subjected to enrichment in minimal medium supplemented with PCE. The bacterial community structure and diversity in the HFBRs were evaluated and compared to samples from the MW1 and MW2, by PCR-DGGE and sequencing of 16S rRNA gene clone libraries. The results revealed the selection of bacterial populations in the HFBR containing inoculum from MW1 (In1) after enrichment, while in the HFBRs containing inoculum from MW2 (In2), the bacterial community structure did not differ from that observed in MW2. Tests of PCE degradation in HFBRs using gas chromatography-mass spectrometry (GC/MS) showed, after 12 hours, an efficiency of 87 % in the PCE degradation in the In1 reactor and 96 % in the In2 reactor. Bacteria from HFBR were isolated and identified by sequencing of 16S rRNA gene, and 4 isolates from In1, similar to Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans and Stenotrophomonas maltophilia, and 7 isolates from In2, similar to Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii and Comamonas sp. were identified. Volatile organic compounds in the reactors with In1 and In2 were analyzed by GC/MS, showing the production of chloroform (TCM) and 1,1,1-trichloroethane (TCA) as PCE degradation products. Consortia composed of bacteria isolated from the In1 (IIn1) and In2 (IIn2) reactors were immobilized and packed in distinct HFBRs to evaluate the potential of specific consortia in PCE degradation. After 12 hours, 92 % of PCE was degraded in reactors with IIn1 and IIn2, with the production of TCM and TCA. Degradation tests using cells suspension were conducted to evaluate the efficiency of each isolate in PCE degradation. Isolate I8 from In2 (I8In2), identified as Comamonas sp., showed 68 % efficiency in the PCE degradation. Assays using inhibitor of monooxygenases cytochrome P-450 (1-aminobenzotriazole) showed that the PCE degradation in HFBRs containing IIn1, IIn2 and I8In2 were dependent of this enzyme. In conclusion, we have identified a new highly efficient PCE degradation pathway, aerobic and mediated by monooxygenases, and isolated bacterial strains that may be used as consortia which immobilized in HFBRs as an efficient alternative in the remediation of PCE contaminated areas.

O tetracloroeteno (PCE), um dos principais contaminantes de águas subterrâneas, é uma molécula recalcitrante, com toxicidade elevada. Processos de biorremediação de água ou solo contaminados com PCE são normalmente limitados pela baixa eficiência de microrganismos sabidamente envolvidos em sua degradação. No entanto, a prospecção de novos microrganismos, mais eficientes na degradação do PCE é uma alternativa para otimizar esses processos. Os objetivos deste estudo foram desenvolver uma técnica de biorremediação utilizando um reator horizontal de leito fixo (RHLF) contendo consórcios de microrganismos eficientes na degradação do PCE, e caracterizar a via de degradação do PCE utilizada pelo consórcio selecionado. Para tanto, amostras de sedimento de dois poços de monitoramento de água foram coletadas de uma metalúrgica com histórico de contaminação com PCE. Os sedimentos foram imobilizados, acondicionados em RHLFs específicos e submetidos a cultivo de enriquecimento em meio mínimo suplementado com PCE. A estrutura das comunidades e a diversidade bacteriana dos RHLFs foram avaliadas e comparadas com as amostras do PM1 e PM2, por PCR-DGGE e sequenciamento de bibliotecas de clones do gene rRNA 16S. Os resultados evidenciaram a seleção de populações bacterianas no RHLF contendo o inóculo do PM1 (In1) após o cultivo de enriquecimento, enquanto no RHLF contendo o inóculo do PM2 (In2), a estrutura da comunidade bacteriana não diferiu daquela observada no PM2. Ensaios de degradação do PCE nos RHLFs, usando cromatografia gasosa associada à espectrometria de massas (CG/EM), mostraram, após 12 horas, uma eficiência de 87 % na degradação do PCE no reator com In1 e 96 % no reator com In2. Foi feito o isolamento e identificação, por sequenciamento do gene rRNA 16S, das bactérias dos RHLFs, sendo identificados 4 isolados do In1, similares a Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans e Stenotrophomonas maltophilia e 7 isolados do In2, similares a Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii e Comamonas sp. Compostos orgânicos voláteis nos reatores com In1 e In2 foram analisados por CG/EM, identificando a produção de clorofórmio (TCM) e 1,1,1-tricloroetano (TCA) como produtos da degradação do PCE. Consórcios formados por bactérias isoladas dos reatores In1 (IIn1) e In2 (IIn2) foram imobilizados e acondicionados em RHLFs distintos para avaliar o potencial dos mesmos na degradação do PCE. Após 12 horas, 92 % do PCE foi degradado nos reatores com IIn1 e IIn2, com produção de TCM e TCA. Testes de degradação usando células em suspensão foram conduzidos para avaliar a eficiência de cada isolado na degradação do PCE. O isolado I8 do In2 (I8In2), identificado como Comamonas sp., teve 68 % de eficiência na degradação do PCE. Ensaios com inibidor de monoxigenases do citocromo P-450 (1-aminobenzotriazole) mostraram que a degradação do PCE nos RHLFs, contendo IIn1, IIn2 e I8In2, foram dependentes dessa enzima. Como conclusão, nós identificamos uma nova via de degradação do PCE altamente eficiente, aeróbia e mediada por monoxigenases e isolamos cepas bacterianas que podem ser usadas como consórcios imobilizados nos RHLFs como uma alternativa eficiente na remediação de áreas contaminadas com PCE. / Tetrachloroethene (PCE), one of the main contaminants of groundwater, is a recalcitrant molecule with high toxicity. Bioremediation processes of water or soil contaminated with PCE are usually limited by the low efficiency of microorganisms known to be involved in its degradation. However, the exploration of new and more efficient microorganisms in the degradation of PCE is an alternative to optimize these processes. The objectives of these studies were to develop a bioremediation technique using horizontal fixed bed reactor (HFBR) containing microbial consortia effective in the PCE degradation, and to characterize the PCE degradation pathway used by the selected consortium. For that, sediment samples of two groundwater monitoring wells were collected from a metallurgical plant with historical of PCE contamination. The sediments were immobilized, packed in specific HFBRs and subjected to enrichment in minimal medium supplemented with PCE. The bacterial community structure and diversity in the HFBRs were evaluated and compared to samples from the MW1 and MW2, by PCR-DGGE and sequencing of 16S rRNA gene clone libraries. The results revealed the selection of bacterial populations in the HFBR containing inoculum from MW1 (In1) after enrichment, while in the HFBRs containing inoculum from MW2 (In2), the bacterial community structure did not differ from that observed in MW2. Tests of PCE degradation in HFBRs using gas chromatography-mass spectrometry (GC/MS) showed, after 12 hours, an efficiency of 87 % in the PCE degradation in the In1 reactor and 96 % in the In2 reactor. Bacteria from HFBR were isolated and identified by sequencing of 16S rRNA gene, and 4 isolates from In1, similar to Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans and Stenotrophomonas maltophilia, and 7 isolates from In2, similar to Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii and Comamonas sp. were identified. Volatile organic compounds in the reactors with In1 and In2 were analyzed by GC/MS, showing the production of chloroform (TCM) and 1,1,1-trichloroethane (TCA) as PCE degradation products. Consortia composed of bacteria isolated from the In1 (IIn1) and In2 (IIn2) reactors were immobilized and packed in distinct HFBRs to evaluate the potential of specific consortia in PCE degradation. After 12 hours, 92 % of PCE was degraded in reactors with IIn1 and IIn2, with the production of TCM and TCA. Degradation tests using cells suspension were conducted to evaluate the efficiency of each isolate in PCE degradation. Isolate I8 from In2 (I8In2), identified as Comamonas sp., showed 68 % efficiency in the PCE degradation. Assays using inhibitor of monooxygenases cytochrome P-450 (1-aminobenzotriazole) showed that the PCE degradation in HFBRs containing IIn1, IIn2 and I8In2 were dependent of this enzyme. In conclusion, we have identified a new highly efficient PCE degradation pathway, aerobic and mediated by monooxygenases, and isolated bacterial strains that may be used as consortia which immobilized in HFBRs as an efficient alternative in the remediation of PCE contaminated areas.

Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme. The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a variety of variety leguminous plants and fungi and is a potent and irreversible inhibitor of succinate dehydrogenase. Nitronate monooxygenase allows N. crassa to overcome the toxicity of propionate-3-nitronate as demonstrated by in vivo studies of the yeast, which showed that the wild-type can grow in the presence of the toxin whereas a knock out mutant that lacks the gene encoding for the enzyme could not. In addition to establishing the physiological role of nitronate monooxygenase, the work presented here demonstrates that the catalytic mechanism of the enzyme involves the formation of an anionic flavosemiquinone intermediate. This intermediate is stabilized by the protonated form of an active site histidine residue (His-196) that acts as an electrostatic catalyst for the reaction as demonstrated by pH studies of the reductive half reaction of the enzyme. Histidine 196 also serves as the catalytic base for the reaction of the enzyme with nitroethane as substrate as revealed through mutagenesis studies in which the residue was replaced with an asparagine. The kinetic implications of branching of reaction intermediates in enzymatic catalysis are also demonstrated through studies of the kinetic isotope effects of nitronate monooxygenase with 1,1-[2H2]-nitroethane as substrate. Finally the use of competitive inhibitors as a probe of enzyme structure will be presented through a study of the inhibition of nitronate monooxygenase with mono-valent inorganic ions. The dissertation will close with unpublished work on the enzyme and concluding remarks concerning the biochemistry and physiology of nitronate monooxygenase.

Epilobium hirsutum L. (Onagraceae), a medicinal plant known as hairy willow herb, has been used by people all around the world for treatment or prevention of inflammation, adenoma, rectal bleeding, menstrual disorders, constipates, and prostate. It contains polyphenolics including steroids, tannins such as gallic, ellagic, and p-coumaric acids and flavonoids such as myricetin, isomyricetin, and quercetin. Polyphenols have been known for their multiple biological health benefits, including antioxidant activities. Viscum album L. (Loranthaceae), a species of mistletoe, contains lectins, polypeptides, mucilage, sugar alcohols, flavonoids, lignans, triterpenes, and phenylallyl alcohols. The leaves and twigs of Viscum album L., taken as tea, have been traditionally used for hypertension, stomachache, diarrhea, diabetes, dysuria and also as analgesic and cardiotonic agent in Anatolia, Turkey. In addition, in Europe, sterile extracts of Viscum album L. are among the most common herbal extracts applied in cancer treatment and have been used as prescription drugs, while in US, considered as dietary supplement. Flavin-containing monooxygenases are FAD-containing phase I enzymes responsible for the oxidation of wide-range of nucleophilic nitrogen, sulfur, phosphorus, and selenium heteroatom-containing drugs such as tamoxifen, v methimazole and imipramine, pesticides, neurotoxins, and other chemicals using NADPH as cofactor. The aim of this study was to determine the in vivo effects of Epilobium hirsutum L. and Viscum album L. (subspecies growing on pine trees-subsp. austriacum (Wiesb.) Vollmann) on FMO activity, mRNA and protein expressions in rat liver. The water extracts of Epilobium hirsutum L. (37.5 mg/kg body weight) and Viscum album L. (10 mg/kg body weight) were injected intraperitonally (i.p) into Wistar albino rats for 9 consecutive days. Following the decapitation, the livers were removed and microsomal fractions were prepared by differential centrifugation. Rat liver microsomal FMO activity using methimazole as substrate, mRNA expression by quantitative Real-Time PCR, and protein expression by Western Blot were determined. The results showed that water extract of Epilobium hirsutum L. has no significant effect on FMO activity / however, it decreased significantly (p&lt / 0.05) FMO3 protein and mRNA expression 27.71% and 1.41 fold, respectively, compared as controls. Water extract of Viscum album L. decreased mRNA (2.56 fold), and protein expressions (27.66%) as well as enzyme activity (19%) of FMO with respect to controls. In conclusion, our current data suggest that the metabolism of xenobiotics including drug molecules by FMO-catalyzed reactions may be altered due to the changes in FMO expression and activity by medicinal plants Epilobium hirsutum L. and Viscum album L.

Peptide a-amidation is known as a signature of bioactivity due to the fact that half of the bioactive peptides found in the nervous and endocrine systems are a-amidated and that most known a-amidated peptides are bioactive. a-Amidated peptides are produced by the oxidative cleavage of glycine-extended precursors. Peptidylglycine a-amidating monooxygenase (PAM) is the only known enzyme responsible for catalyzing this reaction and its sole physiological function is to convert glycine extended prohormones to their a-amidated forms. High levels of PAM are found in certain tissues with no corresponding level of amidated products suggesting the presence of undiscovered a-amidated peptide hormones. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for peptide identification due to its advantages of speed, sensitivity and applicability to complex peptide mixtures. Normally, spectra are interpreted using database search engines. However, database searching is inefficient and ineffective for the identification of endogenous peptide with post-translational modifications (PTM) due to its low identification rate and high demand for computing power. There is a specific mass difference of 58.0055 units between an a-amidated peptide and its corresponding C-terminal glycine-extended precursor. The two peptides will have similar chromatographic retention time and MS/MS fragmentation patterns resulting from the identical amino acids sequences except for relatively the small differences at the C-termini. Based on this, a new LC-MS/MS based strategy for screening for a-amidated peptides was developed. This strategy depends on PAM inhibition and the mass accuracy of mass spectrometry (< 3 ppm). The coexistence of a-amidated peptides and their C-terminal glycine-extended precursors was insured by growing cells in the presence of a PAM inhibitor. After LC-MS/MS, masses and retention times of parent ions were extracted from raw data files and scanned by a script for peptide pairs with similar retention times and a mass difference around 58.0055. Resulting pairs were further validated by comparing their fragmentation patterns in MS/MS spectra. Only peptide pairs that met all three criteria were considered for further interpretation. This reduced the number of MS/MS spectra requiring interpretation by >99% and, thus, enable the manual inspection of MS/MS for the candidate peptide pairs. A total of 13 a-amidated peptides were successfully identified from cultured mouse pituitary AtT-20 cells using this method and a few of these newly identified a-amidated peptides exhibited bioactivity. The adaptability of this strategy to screening for other PTMs is also discussed. Peptidylglycine a-hydroxylating monooxygenase (PHM) is one of PAM domains which can be expressed separately. It is a copper dependent enzyme that catalyzes the first step of the two-step peptide amidation reaction. Removal of the copper ions results in the loss of enzyme catalytic activity. A PHM based a-amidated peptide enrichment method was developed. This method includes two steps. First, cells grown in culture were treated with a PAM inhibitor to effect the cellular accumulation of glycine-extended peptides. In the second step, copper-depleted PHM (apo-PHM) was used to selectively bind glycine-extended peptides present in the cell extract. All other unbound peptides were removed during wash runs. apo-PHM was then reinstated with copper to convert bound glycine-extended peptides to hydroxylated peptides and release them. Hydroxylated product can be converted to a-amidated peptide under basic conditions. Experiments carried out using model glycine extended peptides showed a 40 – 120-fold enrichment using HPLC-fluorometric assay or MALDI-TOF quantification. This method proved successful when working with complex samples like cell extracts. The relative intensity of a known a-amidated peptide mouse joining peptide (mJP) from an AtT-20 extract was dramatically increased after enrichment experiments.

Philosophiae Doctor - PhD / Nitric Oxide (NO) influences a wide range of physiological processes in plants including growth and development, responses to abiotic and biotic stress and pathogen responses. NO binds to the heme group of the mammalian soluble guanylyl cyclase, which activates the enzyme to convert guanosine 5’ triphosphate (GTP) to a second messenger guanosine 3’, 5’ cyclic monophosphate (cGMP). Cyclic GMP further activates other signalling cascades including the regulation of protein kinases, ion gated channels and phosphodiesterases. In plants, a few GCs have been identified and these include AtGC1, AtBRI1, AtWAKL10, and AtPSKR1, however, a GC that contains a heme binding motif that senses NO has yet to be identified. In order to identify such molecules, a search motif based on conserved HNOX domains and the conserved and functionally assigned amino acid residues in the catalytic centres of annotated GCs was designed and used to search the Arabidopsis thaliana proteome. Several candidate molecules were identified including a flavin-containing monooxygenase (FMO)-like protein and the At5g57690 which is currently annotated as a diacylglycerol kinase. FMOs found in bacteria, yeast, and animals are the most important monooxygenases since they are involved in xenobiotic metabolism and variability in drug response. FMOs in plants are implicated in catalysing specific steps in auxin biosynthesis,metabolism of glucosinolates and pathogen defense mechanisms. The human diacylglycerol kinase acts as a lipid kinase that mediates a wide range of biological processes which include cell proliferation, differentiation and turmogenesis. In prokaryotes, the structure of Escherichia coli lipid kinase has been solved however, its function has not yet been demonstrated. So far, the occurrence of the diacylglycerol kinases in plants has not yet been reported, and their structure and function also remain elusive. The domain architecture of the 2 molecules (AtNOGC1 and At5g57690) identified by the HNOX-based search strategy revealed that these molecules contain a GC and a heme-binding motif that is conserved among all known heme-binding proteins.In this study, the role of AtNOGC1, a novel NO binding protein in higher plants was investigated and the results showed that this molecule contains an NO-dependant active GC domain. The sequence was first analysed and the location of the HNOX and the GC motifs highlighted. The protein was then recombinatly expressed as a His-SUMO fusion protein and the purification optimised by a second step of ion exchange chromatography. Electrochemical techniques such as cyclic voltammetry and square wave voltammetry were used to demonstrate the binding of NO and O2 to the AtNOGC1. Electrochemical data revealed that AtNOGC1 has a lower affinity for O2 and a higher affinity for NO, an important signalling molecule in plants.The presence of the GC activity in AtNOGC1 was investigated by conducting GC activity assays in vitro in the presence or absence of NO. The GC activity assays demonstrated that AtNOGC1 can synthesize cGMP from GTP in vitro. It was also noted that NO was required for the maximum activation of AtNOGC1 catalytic activity. NO-activated catalysis resulted in a >2 fold excess of cGMP production compared to an NO-independent GC activity assay. The effect of calcium in regulating the GC activity was also investigated and an increase in cGMP levels was observed however, this was just a preliminary finding that requires further experimentation.3 Homology models for both the FMO-like (AtNOGC1) and the diacylglycerol kinase(At5g57690) were built using Modeller program, and important amino acid residues underlying the heme-binding and GC motifs were identified. Residues corresponding to the motifs, which give signature to AtNOGC1 as an FMO, were also noted. In addition,computational functional prediction also suggested the role of AtNOGC1 in a number of processes which include ion binding and functioning as an FMO.Taken together, these findings suggest that AtNOGC1 is a novel Arabidopsis thaliana hemebinding protein that senses NO with higher affinity than for O2. Though AtNOGC1 is currently annotated as a FMO-like protein, it contains a NO-sensitive GC activity and shares limited sequence similarities with mammalian sGC and the recently identified HNOX domains. Homology modelling strongly suggests that AtNOGC1 and At5g57690 belong to the families of FMOs and diacylglycerol kinases respectively. The domain organisation of AtNOGC1 suggests that more of its functions still remain to be identified. The cloning and characterisation of the At5g57690 gene will provide possible means for further experimentation as well as affording more insights into the exact functions of lipid kinases in plants.