Yfid from E.coli as a Pfl repair protein

P. Kolenko^1,3, C. Doberenz², L. Beyer², G. Sawers², M.T. Stubbs³

¹Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2/1888,
162 06 – Prague 6, Czech Republic

²Institut für Mikrobiologie, Martin-Luther Universität, Kurt-Mothes-Straße 3,
06 120 Halle (Saale), Germany

³Institut für Biochemie und Biotechnologie, Martin-Luther Universität, Kurt-Mothes-Straße 3,
06 120 Halle (Saale), Germany

petr.kolenko@gmail.com

Facultative anaerobic bacteria like E.coli are able to undergo transitions between aerobic and anaerobic environments. The bacteria have mechanisms to adapt their metabolism according to the level of oxygen in the environment [1]. Many of the adaptive mechanisms are still not fully understood.

YfiD protein in E.coli is known as a “quick re-activator” of protein PflB, a glycyl radical enzyme that undergoes oxygenolytic cleavage of the polypeptide chain at the site of the radical. A molecular mechanism of the reactivation of PflB by YfiD was suggested after sequence analysis of both proteins. The proteins share high sequence identity in a roughly sixty amino acid-long C-terminal amino acid stretch [2].

We have cloned, expressed, and purified YfiD and three genetically truncated variants of protein PflB. We have performed analysis of the proteins and their interactions using size exclusion chromatography, ITC, pull-down assays, and NMR. We have also performed crystallization trials and NMR measurements for structural studies.

1. E.W. Trotter, M.D. Rolfe, A.M. Hounslow, C.J. Craven, M.P. Williamson, G. Sanguinetti, R.K. Poole, J. Green, PLoS ONE, 6, (2011), e25501.

2. A.F. Volker Wagner, S. Schultz, J. Bomke, T. Pils, W.D. Lehmann, J. Knappe, Bioch. Biophys. Res. Commun., 285, (2001), 456-462.

This work was supported by the GRK1026 of the Deutsche Forschungsgemeinschaft, and by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. CZ.1.07/2.3.00/30.0029).