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Identification of an amino acid residue that activates OxyR resulting in constitutive activation of target genes in Xanthomonas campestris pv phaseoli

Mongkolsuk, S.; Whanksuk, W.

FASEB Journal 17(4-5): Abstract 634

2003


ISSN/ISBN: 0892-6638
Accession: 035073881

The objective of this study is to characterize mutations in oxyR5 which resulted in the transcriptional regulatory protein being locked in the oxidized form and constitutively activated genes in the OxyR regulon. OxyR5 from a X. campestris pv phaseoli H2O2 resistance mutant has two important mutations G197D and L301R. The protein exists in the oxidized-like form in uninduced cells as judged by the protein ability to activate the ahpC promoter. Analysis of footprint patterns from the DNAseI protection assay indicates that OxyR5 and OxyRG197D bind to the target site in the ahpC promoter as oxidized proteins under reducing conditions. Site-directed mutagenesis of the mutant gene shows that OxyR5 can exist in oxidized-like form, independent of the highly conserved C residues at positions 199 and 208 where in normal OxyR, a disulphide bond between these residues converts the protein from its reduced to the oxidized form. The D197 is absolutely required for the conversion of the protein into the oxidized-like form in uninduced cells. The mutation at D197A gives a protein with similar properties to wild type OxyR. In vivo, the OxyR5 probably lock in oxidized-like form resulting in continuous high levels activation of target genes in OxyR regulon. The research was supported by grants from Chulabhorn Research Institute and NSTDA "Research team strengthening grant" .

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