The amine oxidase family of enzymes has been the center of numerous mechanistic studies because of the medical relevance of the reactions they catalyze. This study describes transient and steady-state kinetic analyses of two flavin amine oxidases, mouse polyamine oxidase (PAO) and human lysine specific demethylase (LSD1), to determine the mechanisms of amine oxidation. PAO is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the oxidation of N1-acetylated polyamines. The pH-dependence of the kcat/Kamine indicates that the monoprotonated form of the substrate is required for catalysis, with the N4 nitrogen next to the site of CH bond cleavage being unprotonated. Stopped-flow spectroscopy shows that the pH-dependence of the rate constant for flavin reduction, kred, displays a pKa of 7.3 with a decrease in activity at acidic pH. This is consistent with an uncharged nitrogen being required for catalysis. Mutating Lys315 to methionine has no effect on the kcat/Kamine-pH profile with the substrate spermine, and the kred value only shows a 1.5-fold decrease with respect to wild-type PAO. The mutation results in a 30- fold decrease in kcat/KO2. Solvent isotope effects and proton inventories are consistent with Lys315 accepting a proton from a water molecule hydrogen-bonded to the flavin N5 during flavin oxidation. Steady-state and transient kinetic studies of para-substituted N,N'-dibenzyl-1,4- diaminobutanes as substrates for PAO show that the kred values for each correlate with the van der Waals volume (VW) and the value. The coefficient for VW is the same at pH 8.6 and 6.6, whereas the p value increases from -0.59 at pH 8.6 to -0.09 at pH 6.6. These results are most consistent with a hydride transfer mechanism. The kinetics of oxidation of a peptide substrate by human lysine specific demethylase (LSD1) were also studied. The kcat/KM pH-profile is bell-shaped, indicating the need for one unprotonated nitrogen next to the site of CH bond cleavage and another protonated nitrogen. The kcat and kred values are equal, and identical isotope effects are observed on kred, kcat, and kcat/KM, indicating that CH bond cleavage is rate-limiting with this substrate.

Pseudomonas aeruginosa ᴅ-arginine dehydrogenase (PaDADH) is a flavin-dependent enzyme. The enzyme catalyzes the oxidative deamination of a broad range of ᴅ-amino acids to their corresponding imino-acids, which are non-enzymatically hydrolyzed to α-keto-acids and ammonia. A46, S45 and I335 residues are located in flexible loops, which form a flask-like substrate-binding pocket. In this study, I335, A46, and S45 were mutated to histidine, glycine, and alanine, respectively and individually, through site-directed mutagenesis, to investigate their role in binding and catalysis in PaDADH. The results showed that A46 and S45 residues participate in the optimal orientation of the substrate α-amino group and I335 modulate the active site flexibility.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--Johns Hopkins University, 1995. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 421-441).

Orientador: Paulo Mazzafera / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T20:42:22Z (GMT). No. of bitstreams: 1 Cesarino_Igor_M.pdf: 984828 bytes, checksum: 754aac1bc4502a9d5eb3102a955408d0 (MD5) Previous issue date: 2009 / Resumo: Uma grande quantidade de genes que codificam flavina monooxigenases (Flavin containing monooxygenases - FMOs) é encontrada em genomas vegetais, embora poucas funções biológicas tenham sido relacionadas com esse grupo de enzimas em plantas. Um importante papel desempenhado por FMOs é a conversão de triptamina em N-hidroxil niptamina, reação catalisada pelas proteínas YUCCA de Arabidopsis thaliana e que constitui o passo limitante da via de síntese de auxina a partir de triptofano. Proteínas similares às YUCCA foram descobertas e caracterizadas em outras espécies vegetais, como OsYUCCA em arroz. FLOOZY em petúnia, ToFZY em tomate e SPIl em milho, todas comprovadamente envolvidas na produção do hormônio citado. Análises da proteína recombinante CaFM08 de Coffea arabica revelou características similares às YUCCA, sugerindo que esta proteína de café é a primeira YUCCA-like descrita para esta espécie e, inclusive, para a família Rubiaceae. CaFM08 apresenta os mesmos motivos protéicos conservados entre FMOs vegetais, e particularmente entre proteínas YUCCA-like. O padrão de expressão espacial do gene que codifica CaFM08 indica possível relação com o desenvolvimento de raízes, folhas e flores de café. Apesar de grandes semelhanças com as proteínas YUCCA, a atividade de N-hidroxilação da triptamina não foi comprovada para CaFM08 recombinante in vitro. Uma análise minuciosa a respeito da funcionalidade de CaFM08 produzida em E. coli deve ser feita antes de descartar a participação desta proteína na síntese de auxina. / Abstract: A large number of genes coding flavin-containing monooxygenases (FMOs) is found in plant genomes, although only few biological functions have been related with these enzymes in plants. An important role for FMOs is the conversion of tryptamine in N-hydroxyl triptamine, catalyzed by the YUCCA protein family in Arabidopsis thaliana. These proteins perform "the rate-limiting step in tryptophan-dependent auxin biosynthesis. Similar YUCCA proteins were discovered and characterized in other plant species, like OsYUCCA in rice, FLOOZY in petunia, ToFZY in tomato and SPIl in maize. All of them are shown to be involved in auxin synthesis. Analysis of the recombinant CaFM08 from Colfea Arabica showed features similar to YUCCA proteins, suggesting that CaFM08 is the first described YUCCA-like protein from coffee and, indeed, from the entire Rubiaceae family. CaFM08 has the same conserved motifs found in other plant FMOs and particulary conserved in YUCCA like proteins. The spatial expression pattern from the CaFM08 coding gene suggests a probable role in the development of roots, leaves and flowers. Although very similar to YUCCA proteins, the CaFM08-mediated convertion of tryptamine in N-hydroxyl tryptamine has not been confirmed in vitro. A further analysis of CaFM08 functionality should address the relation of CaFM08 to auxin production. / Mestrado / Mestre em Biologia Vegetal

L'ubiquinone, ou coenzyme Q, est une molécule lipophile polyisoprényle présente dans toutes les membranes biologiques chez les eucaryotes et composée d'un noyau aromatique actif de façon rédox et d'une chaîne grasse. Elle joue un rôle clef dans la chaîne respiratoire et est un important antioxydant membranaire. Chez l'homme, des pathologies sévères sont associées à des mutations de gènes de la biosynthèse de l'ubiquinone. Chez S. cerevisiæ, la biosynthèse de l'ubiquinone est réalisée par un complexe multiprotéique situé à la membrane interne mitochondriale. Certaines étapes de cette voie de biosynthèse ne sont pas encore connues et très peu ont été caractérisées in vitro. L'étude présentée ici a permis d'améliorer la compréhension de l'étape d'hydroxylation en C-5 à laquelle sont associés Coq6, monooxygénase à flavine, ainsi que Arh1 et Yah1, une adrénodoxine réductase et une adrénodoxine. Nous avons réalisé la première purification de Coq6 de S. cerevisiæ avec son cofacteur flavinique et nous avons démontré in vitro l'existence d'une chaîne de transfert d'électrons du NADPH au FAD de Coq6 via l'homologue humain de Arh1 et Yah1. Les études enzymatiques menées avec différents analogues de substrats synthétisés n'ont pas permis de détecter d'activité enzymatique de Coq6 dans les conditions utilisées. Des études préliminaires de fluorescence nous ont néanmoins permis d'avancer une hypothèse quant au substrat de Coq6, qui n'est pas connu avec certitude. Nous avons également réalisé une caractérisation cinétique de la réduction du FAD de l'homologue humain de Arh1 par le NADH et le NADPH, révélant ainsi son comportement particulier avec le NADPH, notamment en présence de Mg2+. / Coenzyme Q, or ubiquinone, is a lipophilic molecule found in all biological membranes in eukaryotes and composed of a redox active aromatic ring and a polyisoprenyl chain. It is a key electron carrier in the respiratory chain and a very important membrane soluble antioxidant. Severe pathologies in humans are associated with mutations in the ubiquinone biosynthesis genes. In S. cerevisiæ, ubiquinone biosynthesis is done by a multiproteic complex at the inner mitochondrial membrane. Some steps of the ubiquinone biosynthesis are still unknown and very few have been characterized in vitro. This study allowed us to better understand the C-5 hydroxylation step that is associated with Coq6, a flavin monooxygenase, Arh1, an adrenodoxin reductase and Yah1, an adrenodoxin. We achieved the first purification of S. cerevisiæ Coq6 with its flavin cofactor and we demonstrated in vitro the existence of an electron transfer chain from NADPH to Coq6 FAD via Arh1 human homologue and Yah1. Enzymatic studies made with several synthetic substrate analogues did not allow us to detect Coq6 enzymatic activity with the tested conditions. Nevertheless, preliminary fluorescence studies led us to make an assumption about Coq6 substrate which is still not well known. We also carried out a kinetic characterization of the NADPH or NADH reduction of Arh1 human homologue, showing its unusual behavior with NADPH, in particular when Mg2+ is present.

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