abstract: Nature is a master at organizing biomolecules in all intracellular processes, and researchers have conducted extensive research to understand the way enzymes interact with each other through spatial and orientation positioning, substrate channeling, compartmentalization, and more. DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build nanodevices ranging from in vitro small molecule biosensing to sophisticated in vivo therapeutic drug delivery systems and multi-enzyme networks. This thesis focuses on how to use DNA nanostructures as programmable biomolecular scaffolds to arranges enzymatic systems. Presented here are a series of studies toward this goal. First, we survey approaches used to generate protein-DNA conjugates and the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, novel strategies for positioning enzymes on DNA nanoscaffolds has been developed and optimized, including site-specific/ non site-specific protein-DNA conjugation, purification and characterization. Third, an artificial swinging arm enzyme-DNA complex has been developed to mimic substrate channeling process. Finally, we extended to build a artificial 2D multi-enzyme network. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2016

El piridoxal 5'-fosfato es la forma biocatalíticamente activa de la vitamina B6, siendo uno de los cofactores más versátiles de la naturaleza, el cuál tiene un papel central en el metabolismo de aminoácidos. Mientras que la mayoria de microorganismos y plantas pueden sintetizar la vitamina B6 de novo, los mamíferos se ven obligados a obtener uno de sus vitámeros a través de la dieta. La maquinaria biosintética de Escherichia coli es, de lejos, la mejor caracterizada y consiste en cuatro proteínas pdx. PdxJ, también conocida como piridoxina 5'-fosfato sintasa, es la enzima clave en esta via. Cataliza el último paso, la complicada reacción de cierre del anillo entre 1-deoxi-D-xilulosa-5-fosfato y aminoacetona-3-fosfato para formar piridoxina 5'-fosfato. La comparación de secuencias de PdxJ entre espécies revela que existe un alto grado de conservación indicando así la enorme importancia fisiológica de esta enzima.Con el uso de un derivado de mercurio fue posible el resolver la estructura cristalina de la enzima de E. coli por el método del "single isomorphous replacement with anomalous scattering" y el refinar la estructura a 2.0 Å de resolución. El monómero corresponde al plegamiento TIM o barril (_/_)8, con la incorporación de tres hélices extra que median los contactos entre intersubunidades en el octámero. El octámero representa el estado fisiológicamente relevante, que fué observado tanto en el cristal como en solución, y que esta organizado como un tetrámero de dímeros activos. La caracterización de la estructura cristalográfica de la enzima con sustratos, análogos de sustrato y productos unidos permitió la identificación del centro activo y la propuesta de un mecanismo detallado. Los rasgos catalíticos más remarcables son: (1) el cierre del centro activo una vez se han unido los sustratos, de manera que el bolsillo de unión queda aislado del solvente y los intermediarios de la reacción quedan así estabilizados; (2) la existencia de dos sitios de unión de fosfato bien definidos; (3) y un canal de agua que penetra el núcleo del barril _ y permite liberar las moléculas de agua formadas durante la reacción.La cantidad de información presentada debería permitir el diseño de inhibidores de la piridoxina 5'-fosfato sintasa basados en su estructura. Es interesante el destacar que entre las bacterias que contienen el gen pdxJ se encuentran unos cuantos patógenos bien conocidos. La resistencia de bacterias contra antibióticos está aumentando cada vez más, hecho que se está convirtiendo en un auténtico problema. Por este motivo, es necesario el desarrollar medicamentos antibacterianos con un alto grado de especificidad y la piridoxina 5'-fosfato sintasa parece ser una diana muy prometedora. / Pyridoxal 5'-phosphate is the biocatalytically active form of vitamin B6, being one of nature's most versatile cofactors that plays a central role in the metabolism of amino acids. Whereas microorganisms and plants can synthetise vitamin B6 de novo, mammals have to obtain one of the B6 vitamers with their diet. The Escherichia coli biosynthetic machinery is the, by far, best characterised and it consists in four pdx proteins. PdxJ, also referred to as pyridoxine 5'-phosphate synthase, is the key enzyme in this pathway. It catalyses the last step, the complicated ring-closure reaction between 1-deoxy-D-xylulose-5-phosphate and aminoacetone-3-phosphate yielding pyridoxine 5'-phosphate. Sequence comparison of PdxJ from different species revealed a remarkable high degree of conservation indicating the paramount physiological importance of this enzyme.With the use of one mercury heavy-atom derivative, it was possible to solve the crystal structure of the E. coli enzyme by the single isomorphous replacement method with anomalous scattering and to refine the structure at 2.0 Å resolution. The monomer folds as a TIM or (_/_)8 barrel, with the incorporation of three extra helices that mediate intersubunits contacts within the octamer. The octamer represents the physiological relevant state that was observed in the crystal and in solution, and that is organised as a tetramer of active dimers. Characterisation of the enzyme crystal structure with bound substrates, substrate analogues, and products allowed the identification of the active site and the proposal of a detailed reaction mechanism. The most important catalytic features are: (1) active site closure upon substrate binding, in order to isolate the specificity pocket from the solvent und thus stabilise the reaction intermediates; (2) the existence of two well-defined phosphate binding sites; (3) and a water channel that penetrates the _ barrel core and allows the release of waters in the closed state.The amount of information here presented should permit the structure-based design of pyridoxine 5'-phosphate synthase inhibitors. Interestingly, among bacteria that contain the pdxJ gene there are several well-known pathogens. More and more, the bacterial resistance against antibiotics is increasing and therefore becoming a real problem. Thus, it is necessary the development of highly specific antibacterial drugs and pyridoxine 5'-phosphate synthase seems to be a promising novel target.

Bacterial type I restriction-modification systems are composed of three different subunits: one HsdS subunit is required for identification of target sequence and anchoring the enzyme complex on DNA; two HsdM subunits in the methyl-transferase complex serve for host genome modification accomplishing a protective function against self-degradation; two HsdR (or motor) subunits house ATP-dependent translocation and consequent cleavage of double stranded DNA activities. The crystal structure of the 120 kDa HsdR subunit of the Type I restriction-modification system EcoR124I in complex with ATP was recently reported. HsdR is organized into four approximately globular structural domains in a nearly square-planar arrangement: the N-terminal endonuclease domain, the RecA-like helicase domains 1 and 2 and the C-terminal helical domain. The near-planar arrangement of globular domains creates prominent grooves between each domain pair. The two helicase-like domains form a canonical helicase cleft in which double-stranded B-form DNA can be accommodated without steric clash. The helical domain, probably involved in complex assembly, exhibits only a few specific interactions with helicase 2 domain. Molecular mechanism of dsDNA translocation, cleavage and ATP hydrolysis has not been yet structurally investigated. Here we propose a translocation cycle of the restriction-modification system EcoR124I based on analysis of available crystal structures of superfamily 2 helicases, strutural modeling and complementary biochemical characterization of mutations introduced in sites potentially inportant for translocation in the HsdR motor subunit. Also a role of the extended region of the helicase motif III in ATPase activity of EcoR124I was probed.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Three experiments were conducted involving 78 pigs castrated males of commercial line. In the first experiment were used 16 animals with average weight of 23.94  0.63 kg, in order to evaluate the chemical composition and nutrient digestibility, nitrogen balance and determination of digestible and metabolizable energy HSC for pigs . The animals were housed in metabolic cages and distributed in two treatments: control diet and food testing, the latter being composed of 70% of the control diet and 30% of HSC. Each treatment with 8 replicates. Although the FPAM have adequate Chemical Composition has low-energy 1,142 kcal / ME / kg and based on this result rations of other experiments were calculated. Nitrogen balance was affected by the inclusion of HSC. In the second experiment, we used 30 pigs with average weight of 22.73  1.79 kg, with the objective of evaluating increasing levels of HSC in diets for growing and finishing pigs on performance, digestibility of feed, carcass characteristics , color and pH of the meat and bioeconomic evaluation of rations. 5 treatments were used, considering increasing levels of HSC 0; 3; 6; 9:12% and 6 replicates per treatment. Increasing levels of HSC affected the performance by detecting downward linear effect for feed intake increased as the inclusion of the ingredient, reflecting positively a better conversion in the growth phase II. It reiterated that the inclusion of 12% of HSC improves the ether extract digestibility coefficients, neutral detergent fiber and gross energy of the feed. It was found that the inclusion of HSC did not affect carcass characteristics, color and pH of animal meats. There was a decreasing linear effect for all economic variables of the growth phase II, except for the economic efficiency ratio which found increasing linear effect. In the finishing phase was quadratic effect for economic efficiency ratio and average cost ratio of feed consumed and decreasing linear effect for partial gross revenue. In the third experiment, we used 32 pigs live weight of 22.64  0.63 kg for evaluating the inclusion of HSC in diets with or without addition of an enzyme supplement composed of xylanase, glucanase and mannanase on digestibility of diets, blood concentrations of triglycerides and urea, performance, carcass characteristics and bioeconomic evaluation of rations. 4 treatments were used: control diet, 12% inclusion of HSC, 12% inclusion of HSC with reduced energy matrix in 100 kcal / kg with and without addition of enzyme complex with 8 replicates per treatment. Enzyme supplementation did not improve the digestibility of nutrients and energy. The inclusion of the ingredient reduced the level of serum urea in all evaluated phases. There were no treatment effects for growth performance, carcass characteristics and bioeconomic evaluation of rations. The inclusion of 12% of HSC without enzyme supplementation can be an economically viable food alternative in the production of pigs for slaughter. / Foram conduzidos trÃs experimentos envolvendo 78 suÃnos, machos castrados de linhagem comercial. No primeiro experimento foram utilizados 16 animais com peso vivo mÃdio de 23,94Â0,63 kg, com o objetivo de avaliar a composiÃÃo bromatÃlogica e os coeficientes de digestibilidade dos nutrientes, balanÃo de nitrogÃnio e determinaÃÃo da energia digestÃvel e metabolizÃvel FPAM para suÃnos. Os animais foram alojados em gaiolas metabÃlicas e distribuÃdos em 2 tratamentos: raÃÃo controle e raÃÃo teste, sendo esta Ãltima composta por 70% da raÃÃo controle e 30% do FPAM. Cada tratamento com 8 repetiÃÃes. Apesar de o FPAM possuir adequada composiÃÃo bromatÃlogica apresenta baixa concentraÃÃo energÃtica 1.142 kcal/EM/kg e com base nesse resultado foram calculadas as raÃÃes dos demais experimentos. O balanÃo de nitrogÃnio foi prejudicado com a inclusÃo do FPAM. No segundo experimento, foram utilizados 30 suÃnos com peso vivo mÃdio de 22,73Â1,79 kg, com o objetivo de avaliar nÃveis crescentes do FPAM em raÃÃes para suÃnos em crescimento e terminaÃÃo sobre o desempenho, digestibilidade das raÃÃes, caracterÃsticas de carcaÃa, cor e pH das carnes e avaliaÃÃo bioeconÃmica das raÃÃes. Foram utilizados 5 tratamentos, considerando nÃveis crescentes do FPAM 0; 3; 6; 9 e 12% e 6 repetiÃÃes por tratamento. Os nÃveis crescentes do FPAM afetaram o desempenho, detectando-se efeito linear decrescente para o consumo de raÃÃo à medida que aumentou a inclusÃo do ingrediente, refletindo positivamente numa melhor conversÃo na fase de crescimento II. Ratificou-se que a inclusÃo de 12% do FPAM melhora os coeficientes de digestibilidade do extrato etÃreo, fibra em detergente neutro e energia bruta das raÃÃes. Verificou-se que a inclusÃo do FPAM nÃo afetou as caracterÃsticas de carcaÃa, cor e pH das carnes dos animais. Houve efeito linear decrescente para todas as variÃveis econÃmicas da fase de crescimento II, exceto para o Ãndice de eficiÃncia econÃmica onde foi verificado efeito linear crescente. Na fase de terminaÃÃo houve efeito quadrÃtico para Ãndice de eficiÃncia econÃmica e Ãndice de custo mÃdio de raÃÃo consumida e efeito linear decrescente para receita bruta parcial. No terceiro experimento, foram utilizados 32 suÃnos de peso vivo mÃdio de 22,64Â0,63 kg para avaliar a inclusÃo do FPAM em raÃÃes com ou sem adiÃÃo de um suplemento enzimÃtico composto por xilanase, glucanase e mananase sobre a digestibilidade das dietas, concentraÃÃes sanguÃneas de triglicerÃdeos e ureia, desempenho, caracterÃsticas de carcaÃa e avaliaÃÃo bioeconÃmica das raÃÃes. Foram utilizados 4 tratamentos: raÃÃo controle, 12% de inclusÃo de FPAM, 12% de inclusÃo do FPAM com reduÃÃo da matriz energÃtica em 100 kcal de EM/kg com e sem adiÃÃo de complexo enzimÃtico com 8 repetiÃÃes por tratamento. A suplementaÃÃo enzimÃtica nÃo melhorou a digestibilidade dos nutrientes e da energia. A inclusÃo do ingrediente reduziu o nÃvel de ureia sÃrico em todas as fases avaliadas. NÃo foram observados efeito dos tratamentos para o desempenho zootÃcnico, caracterÃsticas de carcaÃa e avaliaÃÃo bioeconÃmica das raÃÃes. A inclusÃo de 12% do FPAM sem suplementaÃÃo enzimÃtica pode ser uma alternativa alimentar economicamente viÃvel na produÃÃo de suÃnos para o abate.

N-methylputrescine oxidase (MPO) is a member of the diamine oxidase (DAO) class of enzymes believed to be responsible for synthesis of the alkaloid nicotine in the roots of Nicotiana tabacum (Mizusaki et al., 1972). A purportedly pure MPO protein from tobacco root culture extracts was used to generate immune antiserum in rabbits (McLauchlan et al., 1993). In an attempt to clone a cDNA encoding MPO, we used this antiserum to screen a tobacco cDNA expression library. Unexpectedly, two previously unreported genes with strong homology to members of a gene family encoding S-adenosylhomocysteine hydrolase (SAHH) in N. sylvestris and a gene encoding SAHH in N. tabacum were cloned instead. SAHH is an enzyme of the S-adenosylmethionine (SAM) recycling pathway, which also includes SAM synthetase (SAMS) and methionine synthase (MS). These results led to the hypothesis of a multi-enzyme complex, or metabolon, of at least one member of the nicotine biosynthesis pathway, i.e., MPO, and at least one member of the SAM recycling pathway, i.e., SAHH, during nicotine biosynthesis. Metabolons are stable noncovalent complexes in cells that ensure sufficient passage of the product of one enzyme reaction to the next enzyme in the pathway via a "channel" without equilibrating with the bulk solution (Ovádi, 1991). My research employed co-immunoprecipitation studies to determine if other SAM recycling enzymes are associated in a complex with MPO and SAHH, as well as Northern and Western blot analyses to determine if the genes encoding SAM recycling pathway enzymes are coordinately regulated during nicotine biosynthesis. Our results indicate that nicotine biosynthesis-inducing conditions result in differential mRNA accumulation patterns of the three enzymes of the SAM recycling pathway, although to different extents. However, protein levels of SAM recycling pathway members do not appear to reflect the differential mRNA accumulation patterns. We have firmly established an association of SAHH and an enzyme with DAO activity, purportedly MPO. If the enzyme is proven to be MPO, then our data would constitute the first documentation of an alkaloid metabolon. Finally, using a degenerate primer PCR approach, we have cloned a 986-bp gene fragment with homology to copper amine oxidases, the class to which MPO belongs. / Master of Science

La diatomée d'eau douce A. formosa peut produire des lipides neutres en plus ou moins grandes quantités en fonction des conditions de culture. Ainsi, nous avons montré par exemple qu'une carence en silice stimule la production de triacylglycérols (TAGs) mais génère une diminution de la biomasse. En revanche, nous avons montré que l'addition de bicarbonate et de phytohormones augmente à la fois la biomasse et la production de TAGs. L'ajout de phytohormones dans les milieux de culture de cette diatomée résulte en une augmentation de l'activité d'enzymes dans les extraits et notamment celles du cycle de Benson-Calvin. Parmi ces enzymes, la GAPDH est une enzyme dont l'activité augmente significativement. Nous avons montré que chez A. formosa, cette enzyme forme un complexe ternaire avec la CP12 et la Férrédoxine NADP Réductase (FNR) et non pas avec la CP12 et la phosphoribulokinase comme chez la plupart des organismes photosynthétiques. La régulation de cette enzyme en est de fait modifiée. La phytohormone, 24-épibrassinolide conduit à une augmentation d'activité de la GAPDH qui résulte de la dissociation du complexe GAPDH-CP12 et la GAPDH n'est plus redox régulée. La GAPDH chez les diatomées est donc régulée par des interactions protéineprotéine. / A. formosa, a freshwater diatom, can produce different amounts of neutral lipids such as triacylglycerols (TAGs) under different growth conditions. We showed that as it is well-known for diatoms, starvation for silica increased the production of TAGs but decreased biomass. However, the addition of bicarbonate or phytohormones into the growth medium increased both biomass and TAGs. Addition of phytohormones increased the activities of enzymes in particular those of the Benson-Calvin cycle. Among the target enzymes of the Benson-Calvin cycle, GAPDH was strongly affected. We purified this enzyme and demonstrated that, in the diatom A. formosa, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Calvin cycle, forms a complex with the small chloroplast protein CP12 and Ferredoxin NADP Reductase (FNR), which is involved in the photochemical phase of photosynthesis. In cells treated with the phytohormone, 24-epibrassinolide, GAPDH was "free", not redox-regulated and not associated anymore with CP12. Therefore GAPDH from this diatom is regulated by protein-protein interaction but the GAPDH/CP12/FNR complex replaces the one formed between GAPDH, CP12 and phosphoribulokinase found in most photoautotrophs.