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Biochemical Characterization of Thermocrispum agreste TheA: A Flavin-Dependent N-hydroxylating EnzymeMena Aguilar, Didier Philippe 26 June 2018 (has links)
N-hydroxylating monooxygenases (NMOs) are Class B flavin-dependent monooxygenases found only in fungi and bacteria. These enzymes catalyze the hydroxylation of nucleophilic primary amines, such as those found in histamine, L-ornithine, L-lysine, and small aliphatic diamines. The hydroxamate moiety produced by this reaction is key for the production of siderophores, small chelating compounds that allow survival in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. Herein we present the characterization of TheA, an NMO from Thermocrispum agreste. The enzyme mechanism was studied using steady state kinetic measurements, thermostability, and stopped flow spectrophotometry assays. Using these techniques, the catalytic rates, substrate binding affinities, thermal stability, and coenzyme specificities were determined. Additionally, NADPH analogues were produced to use as tools to study FAD reduction in NMOs. An unspecific reduction reaction of NADP+ using NaB2H4 yielded [6-2H]-NADPH, [2-2H]-NADPH, and [4-2H]-NADPH. Compound identity was confirmed by mass spectrometry and unidimensional proton nuclear magnetic resonance (NMR). Results presented in this thesis lay the foundation for future studies of NMOs using NADPH analogues. In conjunction, these results will improve the general knowledge and understanding of flavoenzymes, ornithine monooxygenases, and their associated mechanisms. / Master of Science in Life Sciences / Due to the surge of more potent and prevalent microbial pathogens, there is a constant search for new and more specific drugs. One approach is to identify and inhibit enzymes that are key for growth of these pathogens. An example of such enzymes is a group called N-hydroxylating monooxygenases (NMOs) that are key for the production of siderophores, small chelating compounds that allow survival of some fungi and bacteria in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. To develop specific inhibitors of NMOs that can be used as drugs to treat these infections, we first need to understand how these enzymes work. Herein, we characterized a novel NMO from Thermocrispum agrestre. Our results highlight the similarities and differences from previously characterized NMOs. Furthermore, we produced analogues of NADPH, a molecule needed for the mechanism of NMOs. The produced compounds will be used in future studies to understand the step-bystep mechanism of the enzyme. In conjunction, these results will improve the general knowledge and understanding of NMOs and their associated mechanisms and lay the foundation for future studies on the identification of drugs that can be used to treat these diseases.
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Structural and Mechanistic Insights From High Resolution Crystal Structures of the Toluene-4-Monooxygenase Catalytic Effector Protein, NAD(P)H Oxidase and Choline OxidaseLountos, George Themistoclis 28 November 2005 (has links)
X-ray crystallography provides detailed information of the atomic structure of macromolecules that aides in the understanding of their molecular function. In this study, the three-dimensional structures of the Toluene-4-monooxygenase catalytic effector protein (T4moD), NAD(P)H oxidase and choline oxidase were determined. The structures of wild-type and two mutant isoforms of T4moD were solved up to 1.7 resolution. Results from the crystallographic studies indicate that there are significant differences between the X-ray structure and the structure previously solved by NMR. The high-resolution structures have helped to define the potential differences in electrostatic surfaces that may govern the feasibility of protein-protein interactions and also reveal a single, well-defined cavity suitable for toluene binding that has substantial different electrostatic properties among the effector protein family members.
The structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis was determined to 1.8 resolution. The flavoenzyme is of considerable interest as it catalyzes the oxidation of two equivalents of NAD(P)H and reduces one equivalent of oxygen to yield two equivalents of water without releasing hydrogen peroxide from the active site. The structure reveals the presence of a redox active cysteine residue that exists as a sulfenic acid and plays an important mechanistic role by reducing hydrogen peroxide to water. Additionally, a tightly bound ADP molecule was discovered in the enzyme which is hypothesized to play an important role in influencing the dual substrate specificity exhibited by the enzyme.
The structure of choline oxidase from Arthrobacter globiformis was solved to 1.86 resolution. Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions. The structure reveals a cavity within the active site, which is suitable for choline binding. This allows for the identification of the putative binding site for choline and residues involved in substrate-binding and catalysis. Additionally, the structure reveals a highly distorted FAD cofactor that contains a C4a-adduct that is proposed to be either an FAD-C4a-OH or FAD-C4a-O2- complex.
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La méthylation flavine-dépendante d’acides nucléiques : aspects évolutifs, métaboliques, biochimiques et spectroscopiques / Flavin-dependent methylation of nucleic acids : evolutionary, metabolic, biochemical and spectroscopic aspectsSournia, Pierre 14 December 2016 (has links)
La méthylation de l’uridine sur son carbone 5 est apparue au cours de l’évolution sous plusieurs formes. Tout d’abord, les thymidylate synthases permettent la synthèse de novo du dTMP, un précurseur essentiel de l’ADN des trois règnes du vivant. Deux familles de thymidylate synthases sont connues à ce jour : ThyA et la flavo-enzyme ThyX, codées par des gènes hétérologues et ayant des structures et mécanismes réactionnels radicalement différents. En outre, cette méthylation de l’uridine est apparue (probablement plus tard) sous forme de modifications post-transcriptionnelles des ARNt et ARNr. Cette thèse vise à questionner les contraintes évolutives ayant menés indépendament à ces quatres types de méthylation de l’uridine.Une première partie décrit l’identification d’une voie métabolique permetant la complémentation du phénotype d’auxotrophie pour la thymidine par des analogues nucléotidiques chez Escherichia coli. Une approche de biologie synthétique en vue d’établir une voie alternative de biosynthèse du thymidylate a aussi été mise en œuvre. Une technique de sélection de gènes de complémentation du phénotype d’auxotrophie pour la thymidine, issus de mutagénèse aléatoire, a pu être développée. Dans une seconde partie, des études biochimiques et sppectroscopiques ont été réalisées sur la méthyle-transférase flavine-dépendante TrmFO, responsable de la méthylation post-transciptionnelle de l’uridine 54 des ARNt de certains microorganismes.L’implication de certains résidus dans la fixation du substrat a pu être déterminée d’une part, et certains intermédiaires réactionnels potentiels ont été caractérisés spectralement d’autre part. Ces dernières observations s’appuient, en outre, sur des études en cours de spectroscopie résolue en temps et des simulations de dynamique moléculaire afin de mieux comprendre les flavoprotéines en général et les méthyle transférases flavine-dépendantes en particulier. / Enzymes catalyzing the methylation of uridine at its carbon 5 position have appeared independently in different forms across evolution. Thymidylate synthases ThyA and the flavoprotein ThyX catalyze the de novo synthesis of dTMP, an essential DNA precursor in the three domains of life. They are encoded by heterologous genes and have drastically different structures and reaction mechanisms. On the other hand, this uridine methylation is also performed by tRNA and rRNA post-transcriptional modification enzymes.This thesis assesses the question of the evolutionary constraints that have led independently to four kinds of uridine methylation. The first part describes the identification of a metabolic pathway allowing the complementation of thymidine auxotrophy by non-natural nucleotide analogs in Escherichia coli. A synthetic biology approach, aiming to establish an alternative pathway for thymidylate biosynthesis, was also implemented and a selection strategy for thymidine auxotrophy-complementing genes, could be developed.In a second part, biochemical and spectral studies where realised on the flavin-dependent methyltransferase TrmFO, responsible for the post-transcriptional methylation of uridine at the invariant position 54 of tRNA in several microorganisms. The involvement of specific amino acid residues in substrate fixation and in stabilization of potential reaction intermediates was demonstrated. Their spectral characterization supports previously proposed reaction schemes for flavin-dependent thymidylate forming enzymes. These observations are currently being pursued by parallel approaches combining time-resolved spectroscopy and molecular dynamics simulations, aiming to further our understanding of how flavin mediates the transfer of carbon molecules from folate to uracil rings.
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Crystal Structures of Nitroalkane Oxidase: Insights into the Structural Basis for Substrate Specificity and the Catalytic MechanismNagpal, Akanksha 19 July 2005 (has links)
Nitrochemicals are widely used as explosives, biocides and drugs. In addition, 3-nitro-tyrosine and other nitrated protein residues are important markers for many cardiovascular, neurodegenerative, and malignant conditions. Because of the wide presence of the nitrocompounds as toxins, potential nitrogen/carbon sources, and metabolic intermediates, different organisms have evolved to produce enzymes that can biodegrade nitrocompounds. The structural studies of the enzymes, which catalyze the removal of nitro group from nitrochemicals, are of considerable interest for both applied and fundamental reasons. The insights into the reaction mechanism of these enzymes can be used for designing efficient biocatalysts for bioremediation and for developing antibiotics for disease resistant microbes.
Nitroalkane oxidase (NAO) produced by
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Flavin-dependent Enzymes in Natural Product BiosynthesisValentino, Hannah Rachel 31 March 2021 (has links)
Natural products are biologically active metabolites produced by fungi, bacteria, and plants that have an extended application in pharmaceutical and chemical industries. Because of their chemical versatility, flavoenzymes are commonly involved in natural product biosynthetic pathways. This has given rise to the identification of flavoenzymes that are promising candidates for biomedical and biotechnical applications. This dissertation discusses the characterization of three flavoenzymes involved in natural product biosynthesis. The class B flavin-dependent monooxygenases S-monoooxygenase from Allium sativum (AsFMO) and N-hydroxylating monooxygenase from Streptomyces sp. XY332 (FzmM) were studied. Both enzymes perform heteroatom oxidation as part of allicin or fosfazinomycin biosynthesis respectively. AsFMO was predicted to oxidize S-allyl-L-cysteine (SAC) to alliin in allicin biosynthesis. Surprisingly, AsFMO exhibited negligible activity with SAC, and instead was highly active with allyl mercaptan and NADPH. This contradicted the initial proposal and suggested that AsFMO is involved in an alternative path producing allicin directly from allyl mercaptan. FzmM was identified to perform multiple N-oxidations which lead to the formation of a nitro group. FzmM performed a highly coupled and specific reaction with L-aspartate and NADPH to produce nitrosuccinate. Both AsFMO and FzmM followed a kinetic mechanism representative of class B flavin-dependent monooxygenases with a rapid pro-R stereospecific reduction and the formation of a C(4a)-hydroperoxyflavin intermediate during oxidation. In addition, the AsFMO structure was obtained and consisted of two domains for FAD and NADPH binding signature of class B monooxygenases. The biochemical and structural study of the Acinetobacter baumannii siderophore interacting protein (BauF) was also accomplished. This enzyme is essential in acinetobactin mediated iron assimilation and is important for virulence. The characterization of the binding and reduction of acinetobactin-ferric iron complex revealed that BauF is specific for this substrate and does not utilize NAD(P)H as an electron donor. The unique activity and structure of BauF can aid future drug design. / Doctor of Philosophy / Plants, fungi, and bacteria synthesize and excrete unique chemicals called secondary metabolites or natural products. These compounds are used for many applications including dyes, flavorings, fragrances, and medicine. To make natural products, organisms use enzymes to perform complex reactions. Studying the enzymes that are involved in natural product pathways is important for understanding how secondary metabolites are made. Additionally, these enzymes can be engineered to perform reactions relevant to biotechnical applications. Our lab specializes in the study of flavoenzymes which use flavin chemistry for catalysis. Flavin is a yellow coenzyme that contributes to wide array of reactions by performing 1 or 2 electron transfers. This dissertation discussed the characterization of three flavoenzymes. The first enzyme is a S- monooxygenase from Allium sativum (garlic) called AsFMO. Reported here is the kinetic and structural characterization of AsFMO. We demonstrated that AsFMO was cabable of performing an unexpected reaction with allyl mercaptan likely converting it into allicin, the main flavor ingredient of garlic. Secondly, we reported the kinetic characterization of a nitro- forming enzyme termed FzmM. Nitro- formation is a valuable process as nitro- compounds are used in industrial organic synthesis. It was shown that FzmM performs nitro- formation with high efficiency and is specific for the substrate L-aspartate. Lastly, this work described the characterization of the the siderophore-interacting protein from Acinetobacter baumannii, BauF, which was predicted to be involved in iron acqusition. A. baumannii is a serious human pathogen with multidrug resistance, and inhibiting iron acquisition has been shown to prevent its survival. The characterization of the enzymes involved in this pathway is essential for developing new treatments for A. baumannii infection. We report the structure and function of BauF confirming its role in A. baumannii iron uptake and providing information that will aid in future drug design.
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Flavins and Their Analogues as Natural and Artificial CatalystsSichula, Vincent A. 02 March 2011 (has links)
No description available.
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