Wild-type and C26A mutant versions of full-length AnfA were overexpressed in E.coli and purified. Neither protein displayed the spectral features observed in the isolated N-terminal domain, indicating the apo-forms of both proteins had been purified. The wild-type protein was shown to be active in vitro as judged by its ability to activate transcription from the anfH promoter. However C26A AnfA had a 4-5-fold reduced ability to form open complexes at anfH compared to the wild-type protein. A similar reduction in ATPase activity was also observed in the mutant protein while DNA binding to an anfH promoter was the same for both wild-type and mutant proteins.
Thus the apo-form of AnfA is competent to activate transcription indicating that the presence of the Fe-S cluster is not required for activity of the protein. However even in the apo-form of AnfA the cysteine 26 residue is still important for the activity of the protein, implying that the cysteine motif may have a role in regulation of AnfA activity other than as a potential ligand to the Fe-S cluster.
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