A novel method to isolate native NIFB-cofactor

Echavarri Erasun, Calos and Gonzalez, E. and Rubio Herrero, Luis Manuel (2015). A novel method to isolate native NIFB-cofactor. In: "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.

Description

Title: A novel method to isolate native NIFB-cofactor
Author/s:
  • Echavarri Erasun, Calos
  • Gonzalez, E.
  • Rubio Herrero, Luis Manuel
Item Type: Presentation at Congress or Conference (Article)
Event Title: XV Congress of the Spanish Society of Nitrogen Fixation, and the IV Portuguese-Spanish Congress on Nitrogen Fixation
Event Dates: 16/06/2015-18/06/2015
Event Location: León
Title of Book: XV Congress of the Spanish Society of Nitrogen Fixation, and the IV Portuguese-Spanish Congress on Nitrogen Fixation
Date: 2015
Subjects:
Faculty: Centro de Investigación en Biotecnología y Genómica de Plantas (CBGP) (UPM)
Department: Otro
Creative Commons Licenses: Recognition - No derivative works - Non commercial

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Abstract

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.

More information

Item ID: 41846
DC Identifier: http://oa.upm.es/41846/
OAI Identifier: oai:oa.upm.es:41846
Official URL: http://congreso.xvsefin.unileon.es/
Deposited by: Memoria Investigacion
Deposited on: 26 Oct 2016 15:32
Last Modified: 26 Oct 2016 15:32
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