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http://dx.doi.org/10.5352/JLS.2006.16.3.485

Identification of Amino Acids Involved in the Sensory Function of the PrrB Histidine Kinase by Site-directed Mutagenesis  

Kim Yong-Jin (Department of Microbiology, Pusan National University)
Ko In-Jeong (Korea Science Academy)
Oh Jeong-Il (Department of Microbiology, Pusan National University)
Publication Information
Journal of Life Science / v.16, no.3, 2006 , pp. 485-492 More about this Journal
Abstract
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 $3^{rd}$ and $4^{th}$ transmembrane helices and the $2^{nd}$ 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 $2^{nd}$ 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.
Keywords
Histidine kinase; photosynthesis; redox sensing; site-directed mutagenesis; two-romponent system;
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