期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 97, 期 14, 页码 7808-7813出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.97.14.7808
关键词
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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 osmo-sensor 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'-[beta,gamma-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.
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