Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain

Yan Zhu, Ling Qin, Takeshi Yoshida, Masayori Inouye

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Most histidine kinases are bifunctional enzymes having both kinase and phosphatase activities. The cytoplasmic kinase domain of EnvZ, a transmembrane histidine kinase functioning as an osmosensor in Escherichia coli, consists of two distinct functional subdomains: domain A [EnvZc(223- 289)] and domain B [EnvZc(290-450)]. NMR studies demonstrated that domain A consists of a four-helix bundle serving as a dimerization and phosphotransfer domain, and domain B functions as the ATP-binding and catalytic domain. Here we demonstrate that domain A by itself has the phosphatase activity both in vitro and in vivo. This phosphatase activity is Mg2+ dependent but is not activated by ADP, ATP, or adenosine 5'-[β,γ-imido]triphosphate (AMPPNP), each of which may serve as a cofactor for the EnvZ phosphatase activity. Domain B showed a small but distinct effect on the domain A phosphatase activity only in the presence of ADP or AMPPNP. However, when domain B was covalently linked to domain A, dramatic cofactor-dependent enhancement of the phosphatase activity was observed. Extending domain A for another 75 residues at the C terminus or 44 residues at the N terminus did not enhance its phosphatase activity. Substitution mutations at His-243, the autophosphorylation site, demonstrate that the His residue plays an essential role in the phosphatase activity. The so-called X-region mutant L288P that is known to specifically abolish the phosphatase activity in EnvZ had no effect on the domain A phosphatase function. We propose that the EnvZ phosphatase activity is regulated by relative positioning of domains A and B, which is controlled by external signals. We also propose that the His-243 residue participates in both kinase and phosphatase reactions.

Original languageEnglish (US)
Pages (from-to)7808-7813
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number14
StatePublished - Jul 5 2000

All Science Journal Classification (ASJC) codes

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