Memorias de investigación
Ponencias en congresos:
A NOVEL METHOD TO ISOLATE NATIVE NIFB-COFACTOR
Año:2015
Áreas de investigación
  • Ciencias naturales y ciencias de la salud
Datos
Descripción
Shah et al (1994) described NifB-cofactor as the product of the NifB protein after accomplishing its in vitro purification in the Klebsiella pneumonia strain UN1217. Characterization of this small Fe-S cluster revealed its O2-labile nature, greenish-brown appearance, EPR silence, and iron-only metal content. Later work showed that NifB-co was a FeMoco precursor that could be reconstituted in vitro using homocitrate, molybdenum and the NifEN scaffold protein (Curatti et al., 2007). Although the NifB-co structure is not fully understood, it was demonstrated that it comprises -at least- the 6Fe-9S core of FeMo-co coordinated with an interstitial light atom (George et al., 2008) that was later shown to be a carbon. Our inability to obtain fully homogeneous NifB-co preparations may be the reason why NifB-co structure is still eluding us, and it may have to do with sample alterations during the isolation procedure due to the effect of detergents and thiol reducing agents. We have developed a new method to isolate intact NifB-co using a modified K. pneumonia UN1217 strain that overexpresses GST-tagged-NifX as a mean to hijack excess NifB-co. Interestingly, GST-NifX purifications present a greenish-brown colour due the presence of Fe-S cluster(s) which suggest that this strategy may be correct. Ethylene production after NifX-NifB-co reconstitution to FeMoco tested positive. We are currently testing NifX-NifB-co preparations for UV/visible, EPR, Mossbaüer, and protein crystallography to elucidate its structure.
Internacional
Si
Nombre congreso
IV Portuguese-Spanish Congress on Nitrogen Fixation. XV National Meeting of the Spanish Society of Nitrogen Fixation
Tipo de participación
960
Lugar del congreso
Leon
Revisores
Si
ISBN o ISSN
0000000000
DOI
Fecha inicio congreso
16/06/2015
Fecha fin congreso
18/06/2015
Desde la página
96
Hasta la página
96
Título de las actas
XV SEFIN
Esta actividad pertenece a memorias de investigación
Participantes
Grupos de investigación, Departamentos, Centros e Institutos de I+D+i relacionados
  • Creador: Grupo de Investigación: BIOLOGÍA MOLECULAR Y COMPUTACIONAL
  • Centro o Instituto I+D+i: Centro de Biotecnología y Genómica de Plantas, CBGP
  • Departamento: Biotecnología - Biología Vegetal