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Journal of Bacteriology, June 2003, p. 3429-3435, Vol. 185, No. 11
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.11.3429-3435.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cysteine-Scanning Analysis of the Dimerization Domain of EnvZ, an Osmosensing Histidine Kinase

Ling Qin, Shengjian Cai,{dagger} Yan Zhu, and Masayori Inouye*

Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Received 27 November 2002/ Accepted 12 March 2003

EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.

* Corresponding author. Mailing address: Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes La., Piscataway, NJ 08854. Phone: (732) 235-4115. Fax: (732) 235-4559. E-mail: inouye{at}umdnj.edu.

{dagger} Present address: Merck Research Laboratories, Merck & Co., Rahway, NJ 07065.

Journal of Bacteriology, June 2003, p. 3429-3435, Vol. 185, No. 11
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.11.3429-3435.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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