Another four mutants also possess Selleckchem AG-881 point mutations at other positions of the gene (shown in Figure  5). All of those mutations lead to an exchange of one particular amino acid in the expressed protein, two of them which are located in the N-region (position 1,177 and 1,178) lead to the exchange of glutamic acid 393 to lysine or glycin, respectively (Table  7 and Figure  5). Thus, 8 of 15 mutants possess a mutation in the kdpD gene. Figure 5 Sequence of KdpD from V. cholerae . Amino acids labeled in green in the regions H, N, G1, F, G2 are conserved in different species [20]. Labeled in red is threonine 283 which

is exchanged by methionine in the dominant mutations of the resistant strains. Amino acids labeled in blue indicate the positions that are modified in four additional mutants (L73P, P341H, E393K and E393G). A comparison of known protein domains in the database Pfam Protein Families [21] resulted in the localization of the affected amino acid in the dimerization/phosphor acceptor domain. LY3039478 research buy Histidine kinase dimers are formed by parallel association of two domains creating 4-helix bundles; usually these domains contain a conserved histidine residue and are activated via trans-autophosphorylation by the

catalytic domain [22]. They subsequently transfer the phosphoryl group to the aspartic acid acceptor residue of a click here response regulator protein. Based on the comparison of conserved regions in a number of bacterial histidine kinases [20], the localization could be specified more precisely between the H–region and the N-region (Figure 

5). The H-region is the most variable sequence of histidine kinases in bacteria and contains Glutamate dehydrogenase the histidine that is phosphorylated in the signal transduction process. The N-region shuttles the gamma-phosphate from ATP to the histidine residue. The mutated amino acid is localized between the conserved H- and N-region (Figure  5) and thus in a part of the protein that shows high interspecies variation [23], which could explain the specificity of vz0825 against V. cholerae. In the two-component system of signal transduction, the histidine kinase transfers the signal to a response regulator. The V. cholerae protein VC_A0531 is the homolog of KdpD in E. coli, the response regulator of which is KdpE [24]. The signal transduction system KdpABC, regulated by KdpD and KdpE, is part of the osmoregulation machinery in bacteria [15]. Compound vz0825 may exert its mode of action by binding to the histidine kinase KdpD and thereby inhibiting signal transduction. This would lead to a deficient uptake of potassium. If this mechanism leads to the observed reduction of bacterial viability remains to be elucidated. Due to a lack of specific information about the potassium regulation in V. cholerae, we compared our findings with results that have been obtained with E. coli. E.