| Abstrakt: |
The two-component system sensor/response regulator pair, FixL/FixJ, controls the expression of Rhizobium melilotinitrogen fixation (nifand fix) genes in response to changes in oxygen concentration. A truncated version of FixL, FixL∗, is an oxygen-binding hemoprotein kinase that phosphorylates and dephosphorylates the nifand fixgene transcriptional activator, FixJ. Phosphorylation of FixJ is required for optimal transcriptional activation, and anaerobic conditions in vitroresult in a substantial increase in the level of FixJ-phosphate. In this study, site-directed mutagenesis was carried out at histidine residues in FixL∗. Mutant FixL∗ derivatives were purified and analyzed in vitrofor their heme/oxygen binding properties and phosphorylation/dephosphorylation activities. Mutation of histidine 285, the putative autophosphorylation site, to glutamine results in the loss of FixL∗ phosphorylation activities. However, this mutant protein retains a substantial level of FixJ-phosphate dephosphorylation activity. Mutation of histidine 194 to asparagine results in the loss of heme binding and in the failure of FixL∗ to regulate its phosphorylation/dephosphorylation activities in response to changes in oxygen concentration. The FixL∗H194N mutant protein also exhibits an increased FixJ phosphorylation activity under aerobic conditions. This study provides further evidence for the importance of the heme binding domain of FixL∗ in regulating FixJ phosphorylation and dephosphorylation activities in response to oxygen. |
