Identification of amino acid residues important for the function of Agrobacterium tumefaciens Irr protein

Authors

  • Sakkarin Bhubhanil,

    1. Applied Biological Sciences, Chulabhorn Graduate Institute, Bangkok, Thailand
    2. Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of Education, Bangkok, Thailand
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  • Nantaporn Ruangkiattikul,

    1. Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand
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  • Phettree Niamyim,

    1. Applied Biological Sciences, Chulabhorn Graduate Institute, Bangkok, Thailand
    2. Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of Education, Bangkok, Thailand
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  • Jareeya Chamsing,

    1. Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of Education, Bangkok, Thailand
    2. Environmental Toxicology, Chulabhorn Graduate Institute, Bangkok, Thailand
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  • Patchara Ngok-ngam,

    1. Applied Biological Sciences, Chulabhorn Graduate Institute, Bangkok, Thailand
    2. Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of Education, Bangkok, Thailand
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  • Rojana Sukchawalit,

    Corresponding author
    1. Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of Education, Bangkok, Thailand
    2. Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand
    • Applied Biological Sciences, Chulabhorn Graduate Institute, Bangkok, Thailand
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  • Skorn Mongkolsuk

    1. Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of Education, Bangkok, Thailand
    2. Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand
    3. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
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Correspondence: Rojana Sukchawalit, Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand. Tel.: +66 02 5740622, ext. 3802; fax: +66 02 5742027; e-mail: rojana@cri.or.th

Abstract

The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (IrrAt) were investigated. Several IrrAt mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of IrrAt. Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of IrrAt. H94 is essential for the iron responsiveness of IrrAt. Furthermore, the IrrAt mutant proteins showed differential abilities to complement the H2O2-hyper-resistant phenotype of an irr mutant.

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