Site-directed mutagenesis of the response regulator DmsR for the dmsCBA operon expression in Rhodobacter sphaeroides f. sp. Denitrificans: An essential residue of proline-130 in the linker.
DmsR protein is a member of the OmpR response regulator subfamily that activates the transcription of the dmsCBA operon in Rhodobacter sphaeroides f. sp. denitrificans. By site-directed mutagenesis some functional amino acid residues were investigated in DmsR, which consists of the N-terminal regulatory and the C-terminal DNA-binding domains and the linker connecting the two domains. The substitution of P130S in the linker caused decreases of both DNA-binding and transcriptional activator activities. Introducing additional substitutions of R129P or D131P to the DmsR-P130S derivative recovered both activities, demonstrating necessity of proline residue at one of the positions 129-131 in the linker. Substitutions of D12A, D55A, and K104M, at residues conserved in the phosphorylation region, caused no production of DMSO reductase, but retained DNA-binding ability, suggesting that unphosphorylated DmsR also has high affinity to its target nucleotide sequence of DNA. Substitutions in the C-terminal domain suggested the presence of a winged helix-turn-helix structure observed in the DNA-binding domain of the Escherichia coli OmpR.
Yamamoto
I
I
Department of Biological Science, Faculty of Science, Hiroshima University, Higashi-Hiroshima, Japan. iyamamo@ipc.hiroshima-u.ac.jp
Takamatsu
K
K
Ohshima
Y
Y
Ujiiye
T
T
Satoh
T
T
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D015151:Immunoblotting; D007506:Iron-Sulfur Proteins; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010088:Oxidoreductases / Q000096:biosynthesis; D010766:Phosphorylation; D010957:Plasmids; D011392:Proline / Q000737:chemistry; D012242:Rhodobacter sphaeroides / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C107311:DmsR protein, Rhodobacter sphaeroides; D007506:Iron-Sulfur Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D011392:Proline; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase