title: Complexation precedes phosphorylation for two-component regulatory system FixL/FixJ of Sinorhizobium meliloti.
abstract: The FixL/FixJ two-component regulatory system of Sinorhizobium meliloti controls the expression of nitrogen fixation genes in response to O2. When phosphorylated, the transcription factor FixJ binds to the nifA and fixK promoters in S. meliloti and induces expression of the corresponding genes, both of which encode key transcription activators. Phosphorylation of FixJ has been proposed to occur via the following cascade. The sensor kinase FixL reacts with ATP independently of FixJ, transferring a phosphoryl group to one of its own histidine residues. Dissociation of O2 from a heme-binding PAS domain in FixL greatly accelerates the rate of this autophosphorylation. The phosphoryl group is rapidly transferred from phospho-FixL to an aspartate residue on FixJ. The resulting phospho-FixJ is short-lived, due to a FixL-catalyzed hydrolysis of the aspartyl phosphate. Here, we show that phosphorylation of FixLJ, i.e. the complex of FixL with FixJ, is at least tenfold faster than the phosphorylation of FixL without FixJ. We further show that a phospho-FixJ phosphatase, thought to reside in FixL, is absent from this complex. These results indicate that FixLJ reacts with ATP as a unit and much more efficiently than FixL alone, and that autophosphorylation and phosphoryl transfer do not occur independently, in sequence, but rather in a closely coupled processive reaction. These findings highlight the possible influence of synergistic interactions of the regulatory components in two-component-system signal transduction.
authors.affiliation: Departments of Biochemistry, Plant Biology, and the Plant Biotechnology Center, The Ohio State University, 1060 Carmack Road, Columbus, OH 43210-1002, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Tuckerman
authors.forename: G
authors.initials: G
authors.lastname: Gonzalez
authors.forename: M A
authors.initials: MA
authors.lastname: Gilles-Gonzalez
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006420:Hemeproteins / Q000378:metabolism*; D000071677:Histidine Kinase; D007700:Kinetics; D046911:Macromolecular Substances; D009586:Nitrogen Fixation; D010100:Oxygen / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006420:Hemeproteins; D046911:Macromolecular Substances; C069289:FixJ protein, Bacteria; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010749:Phosphoprotein Phosphatases; D010100:Oxygen