A novel method to isolate native NIFB-cofactor

Echavarri Erasun, Calos; Gonzalez, E. y Rubio Herrero, Luis Manuel (2015). A novel method to isolate native NIFB-cofactor. En: "XV Congress of the Spanish Society of Nitrogen Fixation, and the IV Portuguese-Spanish Congress on Nitrogen Fixation", 16/06/2015-18/06/2015, León. p. 1.

Descripción

Título: A novel method to isolate native NIFB-cofactor
Autor/es:
  • Echavarri Erasun, Calos
  • Gonzalez, E.
  • Rubio Herrero, Luis Manuel
Tipo de Documento: Ponencia en Congreso o Jornada (Artículo)
Título del Evento: XV Congress of the Spanish Society of Nitrogen Fixation, and the IV Portuguese-Spanish Congress on Nitrogen Fixation
Fechas del Evento: 16/06/2015-18/06/2015
Lugar del Evento: León
Título del Libro: XV Congress of the Spanish Society of Nitrogen Fixation, and the IV Portuguese-Spanish Congress on Nitrogen Fixation
Fecha: 2015
Materias:
Escuela: Centro de Investigación en Biotecnología y Genómica de Plantas (CBGP) (UPM)
Departamento: Otro
Licencias Creative Commons: Reconocimiento - Sin obra derivada - No comercial

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Resumen

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.

Más información

ID de Registro: 41846
Identificador DC: http://oa.upm.es/41846/
Identificador OAI: oai:oa.upm.es:41846
URL Oficial: http://congreso.xvsefin.unileon.es/
Depositado por: Memoria Investigacion
Depositado el: 26 Oct 2016 15:32
Ultima Modificación: 26 Oct 2016 15:32
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