title: Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5.
abstract: Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme:protein, 1:1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O(2), and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O(2) binding site at the heme distal side. The O(2) dissociation rate constant, 0.10 s(-1), was substantially increased up to 8.0 s(-1) upon Y45L mutation. The resonance Raman frequencies representing ν(Fe-O2) (559 cm(-1)) and ν(O-O) (1149 cm(-1)) of the Fe(II)-O(2) complex of Y45F mutant AfGcHK were distinct from those of the wild-type protein (ν(Fe-O2), 557 cm(-1); ν(O-O), 1141 cm(-1)), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kitanishi
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ishimori
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan.
authors.forename: Jotaro
authors.initials: J
authors.lastname: Igarashi
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan. Electronic address: shimizu@tagen.tohoku.ac.jp.
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000071677:Histidine Kinase; D020125:Mutation, Missense; D009231:Myxococcales / Q000201:enzymology* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D006418:Heme; D011494:Protein Kinases; D000071677:Histidine Kinase; D010100:Oxygen