Producción y caracterización molecular de enzimas relacionadas con nad+ y sus intermedios avanzados

En la Tesis Doctoral titulada "Produccion y caracterizacion molecular de enzimas relacionadas con NAD+ y sus intermedios avanzados", los objetivos fueron: el desarrollo de un metodo enzimatico para la obtencion de nicotinamida mononucleotido (NMN) y nicotinamida ribosido (NR); el diseno de un metodo de cribado funcional para la obtencion de nicotinamidasas/pirazinamidasas en librerias metagenomicas/ poligenomicas; la aplicacion del metodo de cribado desarrollado para encontrar nuevas nicotinamidasas/pirazinamidasas metagenomicas en ambientes extremos; el analisis del genoma de la trufa negra para asignarle actividad nicotinamidasa/pirazinamidasa a una proteina no caracterizada en base a su caracterizacion funcional; y la caracterizacion bioquimica y estructural de una proteina no caracterizada de Oceanobacillus iheyensis como un nuevo macrodominio bacteriano. Para obtener NMN y NR puros, una difosfatasa de NAD+ y una 5'-nucleotidasa bacterianas se clonaron y purificaron. La primera enzima se utilizo para convertir NAD+ en NMN y AMP, siendo este ultimo separado del NMN por cromatografia de intercambio ionico. El NMN obtenido se transformo en NR con la 5'-nucleotidasa. Asimismo, ambos compuestos fueron testados y comparados con la fuente comercial de NMN en dos modelos celulares, encontrando, por primera vez, un mejor incremento de los niveles de NAD+ en uno de los dos modelos tratado con nuestros compuestos. Para el cribado funcional de nicotinamidasas/pirazinamidasas se desarrollaron dos metodos basados en la reaccion de los productos de reaccion de las nicotinamidasas (el acido pirazinoico y el acido nicotinico) con el sulfato amonico ferroso (AFS) y el nitroprusato de sodio (SNP). Una vez establecidas las condiciones optimas de los ensayos, se estudio una libreria de fosmidos poligenomica, encontrando varios clones positivos con el metodo del AFS. La deteccion de actividad de forma cuantitativa con el metodo del SNP nos permitio descubrir la primera nicotinamidasa con actividad balanceada frente a nicotinamida y pirazinamida. Ademas, su caracterizacion bioquimica hizo posible el desarrollo de un metodo para el cribado de inhibidores de nicotinamidasas. Utilizando el metodo descrito anteriormente se descubrio la primera nicotinamidasa hipertermofila procedente de una bacteria no clasificada (UbNic), con una temperatura optima de 90 °C y un amplio pH optimo. La enzima mostro una de las eficiencias cataliticas mas altas entre las nicotinamidasas de bacterias no patogenas. Asimismo, su secuencia de union a metal fue descrita y catalogada en un subgrupo no descrito hasta ahora. Con el conocimiento adquirido en el campo de las nicotinamidasas, se realizo un analisis bioinformatico con el fin de identificar un nuevo gen de trufa como una posible nicotinamidasa. La presencia del gen en el micelio del hongo se demostro con el metodo del AFS descrito anteriormente. Despues de su expresion y purificacion recombinante, se realizo una caracterizacion bioquimica de la proteina, la cual mostro una clara preferencia por nicotinamida frente a pirazinamida. Asimismo, la enzima mostro un patron de inhibicion caracteristico frente a los diferentes aldehidos probados. Finalmente, la caracterizacion funcional y estructural del macrodominio de Oceanobacillus iheyensis nos permitio profundizar en el conocimiento de su mecanismo de catalisis y union a sustrato. Las estructuras cristalinas de los mutantes D40A, N30A y G37V, asi como las obtenidas con MES, ADP-ribosa y ADP, permitieron la identificacion de cinco moleculas de agua encargadas de la union a sustrato. Asimismo, se demostro que el cierre del lazo ?6-?4 es esencial para el reconocimiento del pirofosfato y la orientacion de la ribosa distal. Ademas, se encontro que OiMacroD cataliza tanto la hidrolisis de O-acetil-ADP-ribosa como la eliminacion de residuos de ADP-ribosa de proteinas ribosiladas. Finalmente, el estudio del mutante G37V demostro la participacion de una molecula de agua coordinada con el sustrato que ayuda a mantener la correcta conformacion del mismo. In the PhD Thesis entitled "Production and molecular characterization of NAD+-related enzymes and their advanced intermediates", the objectives were: the development of an enzymatic synthesis method in order to obtain pure nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR); the design of a functional screening method to discover new nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries; the application of the developed functional screening to find new extremophile metagenomic nicotinamidases/pyrazinamidases; the analysis of the black truffle genome to assign a putative protein as a nicotinamidase/pyrazinamidase based on its functional characterization; and the biochemical and structural characterization of a hypothetical protein from Oceanobacillus iheyensis as a MacroD-like macrodomain. To obtain pure NMN and NR, a bacterial NAD+-diphosphatase and 5'-nucleotidase were cloned and purified. The first enzyme was able to convert NAD+ into NMN and AMP, being the latter compound separated from NMN by ion exchange chromatography. The NMN obtained was fully transformed into NR by the 5'-nucleotidase. Furthermore, both compounds were tested and compared with commercial NMN in two cellular models, finding not only the same NAD+ increase in one of the cell types but also, for the first time, a higher increment in the NAD+ levels of the other cellular model treated with our enzymatic compounds. For the functional screening of nicotinamidases/pyrazinamidases, two new whole-cell methods were developed using the chemical property of the products formed in the enzymatic reaction catalyzed by nicotinamidases (pyrazinoic or nicotinic acids) to form colored complexes with the stable iron salts ammonium ferrous sulfate (AFS) or sodium nitroprusside (SNP). After the optimization of the conditions, a fosmid polygenomic library was screened, discovering several positive clones with the AFS method. Their quantitative re-screening with the SNP method allowed us to discover the first nicotinamidase with balanced catalytic efficiency towards nicotinamide and pyrazinamide. Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of nicotinamidase inhibitors by the naked eye. Using the previously developed method, together with bioinformatics, the first hyperthermophilic nicotinamidase from an unclassified bacterium (UbNic) was found, with an optimum temperature of 90 °C and a broad optimum pH. The enzyme showed one of the highest catalytic efficiencies among non-pathogenic bacterial nicotinamidases. Furthermore, the sequence of the metal binding site revealed that UbNic and its related sequences belong to a subgroup not described so far. With the knowledge acquired in the field of nicotinamidases, a bioinformatic analysis was performed to identify a new gene from truffle as a putative nicotinamidase. Its presence in the mycelium of the fungus was demonstrated with the AFS method developed before. After its recombinant expression and purification, a biochemical characterization of the protein was carried out, finding that this nicotinamidase has a clear preference for its natural substrate (NAM) than for pyrazinamide. The enzyme also had a unique inhibition pattern with different aldehydes. Finally, the functional and structural characterization of the macrodomain from Oceanobacillus iheyensis allowed us to shed light on its substrate binding and catalysis. The crystal structures of D40A, N30A and G37V mutants, and those with MES, ADP-ribose and ADP bound, led us to the identification of five fixed water molecules that play a significant role in substrate-binding. Furthermore, the closure of the ?6-?4 loop was revealed as essential for pyrophosphate recognition and distal ribose orientation. In addition, a novel structural role for residue D40 was identified. Moreover, it was revealed that OiMacroD catalyzes the hydrolysis of O-acetyl-ADP-ribose and also reverses protein mono-ADP-ribosylation. Finally, mutant G37V supports the participation of a substrate-coordinated water molecule in catalysis that helps to select the proper substrate conformation.