title: Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain.
abstract: 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 Mg(2+) 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.
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: L
authors.initials: L
authors.lastname: Qin
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: M
authors.initials: M
authors.lastname: Inouye
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine; D000071677:Histidine Kinase; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D006639:Histidine; D011494:Protein Kinases; D000071677:Histidine Kinase; C057843:envZ protein, E coli; D010749:Phosphoprotein Phosphatases