Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus

Authors

Heidi G. Standke, Chapman University
Lois Kim, Chapman University
Cedric P. Owens, Chapman UniversityFollow

Document Type

Article

Publication Date

10-3-2023

Abstract

NifA is a σ⁵⁴ activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein’s central AAA⁺ domain. In this work, we examine if an additional Cys pair that is part of a C(X)5 C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues’ redox state may directly influence the DNA binding domain’s DNA binding affinity and/or alter the protein’s oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner.

Comments

This article was originally published in Protein Journal, volume 42, issue 6, in 2023. https://doi.org/10.1007/s10930-023-10158-w

Recommended Citation

Standke, H.G., Kim, L. & Owens, C.P. Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus. Protein J 42, 802–810 (2023). https://doi.org/10.1007/s10930-023-10158-w

Copyright

The authors

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.