Truncation of amino acids 12-128 causes deregulation of the phosphatase activity of the sensor kinase KdpD of Escherichia coli.

The kdpFABC operon, which encodes the structural genes for the high affinity K+ transport complex KdpFABC, is regulated by the sensor kinase KdpD and the response regulator KdpE. KdpD is a bifunctional enzyme catalyzing the autophosphorylation by ATP and the dephosphorylation of the corresponding response regulator KdpE. Here, we demonstrate that the phosphatase activity of KdpD is dependent on ATP, whereas GTP, ITP, CTP, ADP, and GDP have no effect. The phosphatase activity requires only ATP binding, because nonhydrolyzable analogs (adenosine-5'-[gamma-thio]triphosphate and adenosine-5'-[beta,gamma-imido]triphosphate) work as well. However, KdpD proteins missing amino acids 12-128 are characterized by a phosphatase activity that is independent of ATP. These proteins are still able to respond to K+ starvation, but an increase in osmolarity is no longer sensed. Comparison of different KdpD sequences reveals a conserved motif in this amino acid region that is very similar to a classical ATP-binding site (Walker A motif). Replacement of the conserved Gly37, Lys38, and Thr39 residues in the consensus ATP-binding sequence results in a KdpD protein that causes a kdpFABC expression pattern comparable with that seen with KdpD proteins missing amino acids 12-128. However, in vitro phosphatase activity is comparable with that of wild-type KdpD. These results suggest that amino acids 12-128 of KdpD are important for its activity and that an additional ATP-binding site in the N-terminal region seems to be involved in modulation of the phosphatase activity.