Predicción bioinformática de residuos de aminoácidos importantes en la dimerización de la enzima nitrato reductasa de Penicillium purpurogenum
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2010
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
La asimilación de nitrato en hongos involucra su reducción por la enzima nitrato
reductasa (NR) formando como producto nitrito el cual es reducido a amonio por la enzima
nitrito reductasa (NiR). La NR eucarionte tiene 3 dominios principales, los cuales son el
dominio de unión a molibdopterina (MoCo), citocromo y el dominio de unión a Flavín
adenín dinucleótido (FAD). Se ha reportado que las NR de otros eucariontes distintos a los
hongos forman homodímeros, y aunque no se han podido hacer estudios precisos sobre la
zona de interface entre los monómeros del dímero, se sugiere que las interacciones tipo
puente de hidrógeno y puente salino son responsables de la estabilización del dímero
MoCo. Esta posibilidad no ha sido analizada en NR fúngicas. Por esta razón, en esta tesis se
secuenció el gen de la NR (niaD) en Penicillium purpurogenum y se caracterizó in sílico las
posibles interacciones de la interfase del dímero. El gen se amplificó y secuenció mediante
técnicas basadas en PCR. El promotor contiene cajas de interés como secuencias GATA
(regulador del metabolismo del nitrógeno en hongos). Con la secuencia aminoacídica
deducida se realizó modelamiento de la proteína NR en sus 3 dominios (MoCo, citocromo y
FAD) y se sometió el modelo del dominio MoCo a acoplamiento proteína-proteína para
analizar in sílico la interfase de dimerización con mutaciones de posibles residuos
involucrados. Adicionalmente, se generó un sistema de complementación de P.
purpurogenum basado en el gen niaD. Se obtuvieron mutantes niaD· mediante irradiación
UV, y posterior selección utilizando clorato y diferentes fuentes de nitrógeno. Una vez
obtenidas las mutantes fenotípicas niaD·, se complementó la cepa mutante con un plásmido
conteniendo el gen niaD homólogo de P. purpurogenum. Este sistema permitirá, a futuro,
comprobar in vivo si los residuos propuestos en esta tesis son importantes en el papel
fisiológico de NR en este hongo.
Nitrate assimilation in fungi involves nitrate reduction by the enzyme nitrate reductase (NR) forming nitrite, which in turn is reduced to ammonium by the enzyme nitrite reductase (NIR). Eukaryotic NR has 3 main domains, named molybdopterin binding domain (MoCo), cytochrome and flavin adenine dinucleotide binding domain FAD. It has been reported that NR from cukaryotes different from fungi form homodimers. Although detailed studies of the zone of interface between the monomers of the dimer have not been carried out, it has been suggested that hydrogen bonds and salt bridges may be responsible for dimer stabilization in the MoCo domain. This possibility has not been explored for fungal NR. For this reason, in this thesis the NR gene (niaD) from Penicillium purpurogenum was sequenced, its structure was modeled at the dimer interface were characterized in silico. The gene was amplified and sequenced using PCR-based techniques. Its promoter contains sequences for the putative binding of transcriptional regulators such as GATA (regulator of nitrogen metabolism in fungi). The deduced amino acid sequence was used for the modeling of the 3 domains of NR protein (MoCo, cytochrome and FAD). To analyze the dimerization interface, the MoCo, this domain was submitted to protein protein docking in silico using virtual mutations on some residues. In addition, a complementation system for P. purpurogenum based on the nia gene was generated. The niat mutants were obtained by UV irradiation, and subsequent selection using chlorate and nitrogen sources. Finally, one of the niab mutants was complemented with a plasmid containing the homologous gene nia from P. purpurogerum. In future, work this system will allow an in vivo check if amino acids residues that are proposed in this thesis have a physiological role in P. purpurogenum NR.
Nitrate assimilation in fungi involves nitrate reduction by the enzyme nitrate reductase (NR) forming nitrite, which in turn is reduced to ammonium by the enzyme nitrite reductase (NIR). Eukaryotic NR has 3 main domains, named molybdopterin binding domain (MoCo), cytochrome and flavin adenine dinucleotide binding domain FAD. It has been reported that NR from cukaryotes different from fungi form homodimers. Although detailed studies of the zone of interface between the monomers of the dimer have not been carried out, it has been suggested that hydrogen bonds and salt bridges may be responsible for dimer stabilization in the MoCo domain. This possibility has not been explored for fungal NR. For this reason, in this thesis the NR gene (niaD) from Penicillium purpurogenum was sequenced, its structure was modeled at the dimer interface were characterized in silico. The gene was amplified and sequenced using PCR-based techniques. Its promoter contains sequences for the putative binding of transcriptional regulators such as GATA (regulator of nitrogen metabolism in fungi). The deduced amino acid sequence was used for the modeling of the 3 domains of NR protein (MoCo, cytochrome and FAD). To analyze the dimerization interface, the MoCo, this domain was submitted to protein protein docking in silico using virtual mutations on some residues. In addition, a complementation system for P. purpurogenum based on the nia gene was generated. The niat mutants were obtained by UV irradiation, and subsequent selection using chlorate and nitrogen sources. Finally, one of the niab mutants was complemented with a plasmid containing the homologous gene nia from P. purpurogerum. In future, work this system will allow an in vivo check if amino acids residues that are proposed in this thesis have a physiological role in P. purpurogenum NR.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Palabras clave
Bioinformática Investigaciones