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KMID : 0613820060160030485
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Journal of Life Science
2006 Volume.16 No. 3 p.485 ~ p.492
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Identification of Amino Acids Involved in the Sensory Function of the PrrB Histidine Kinase by Site-directed Mutagenesis
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Kim Yong-Jin
Ko In-Jeong
Oh Jeong-Il
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Abstract
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The PrrBA two-component system is one of the major regulatory systems that control expression of photosynthesis genes in response to changes in oxygen tension in the anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides. The system consists of the PrrB histidine kinase and the PrrA response regulator. The N-terminal transmembrane domain of PrrB serves as a signal-sensing domain and comprises six transmembrane helices forming three periplasmic loops and two cytoplasmic loops. The 3rd and 4th transmembrane helices and the 2nd periplasmic loop were suggested to play a crucial role in redox-sensory function. In this study we demonstrated that mutations of Asp-90, Gln-93, Leu-94, Leu-98, and Asn-106 in the 2nd periplasmic loop and its neighboring region led to severe defects in PrrB sensory function, indicating that these amino acids might be related to the redox-sensing function of PrrB. The mutant forms (D90E, D90N, and D90A) of PrrB were heterologously overexpressed in Escherichia coli, purified by means of affinity chromatography and their autokinase activities were comparatively assessed. The D90N form of PrrB was shown to possess higher autokinase activity than the wild-type form of PrrB, whereas the D90E form of PrrB displayed lower autokinase activity than the wild-type form of PrrB. The D90A mutation led to the loss of PrrB autokinase activity.
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KEYWORD
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Histidine kinase, photosynthesis, redox sensing, site-directed mutagenesis, two-component system
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