Domain characterization of Bacillus subtilis GabR, a pyridoxal 5'-phosphate-dependent transcriptional regulator.
Bacillus subtilis GabR is a transcriptional regulator consisting of a helix-turn-helix N-terminal DNA-binding domain, a pyridoxal 5'-phosphate (PLP)-binding C-terminal domain that has a structure homologous to aminotransferases, and a linker of 29 amino acid residues. In the presence of γ-aminobutyrate (GABA), GabR activates the transcription of gabT and gabD, which encode GABA aminotransferase and succinate semialdehyde dehydrogenase, respectively. We expressed N-terminal and C-terminal domain fragments (named N'-GabR and C'-GabR) in Escherichia coli cells, and obtained N'-GabR as a soluble monomer and C'-GabR as a soluble dimer. Spectroscopic studies suggested that C'-GabR contains PLP and binds to d-Ala, β-Ala, d-Asn and d-Gln, as well as GABA, although the intact GabR binds only to GABA. N'-GabR does not bind to the DNA fragment containing the GabR-binding sequence regardless of the presence or absence of C'-GabR. A fusion protein consisting of N'-GabR and 2-aminoadipate aminotransferase of Thermus thermophilus bound to the DNA fragment. These results suggested that each domain of GabR could be an independent folding unit. The C-terminal domain provides the N-terminal domain with DNA-binding ability via dimerization. The N-terminal domain controls the ligand specificity of the C-terminal domain. Connection by the linker is indispensable for the mutual interaction of the domains.
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Frou-Chou, Chikusa, Nagoya, Aichi 464-8601, Japan and.
Keita
K
Okuda
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Frou-Chou, Chikusa, Nagoya, Aichi 464-8601, Japan and.
Tomokazu
T
Ito
Department of Biomolecular Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan.
Masaru
M
Goto
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Frou-Chou, Chikusa, Nagoya, Aichi 464-8601, Japan and.
Takashi
T
Takenaka
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Frou-Chou, Chikusa, Nagoya, Aichi 464-8601, Japan and.
Hisashi
H
Hemmi
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Frou-Chou, Chikusa, Nagoya, Aichi 464-8601, Japan and yosimura@agr.nagoya-u.ac.jp.
Tohru
T
Yoshimura
D000612:4-Aminobutyrate Transaminase / Q000737:chemistry / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D019077:Helix-Turn-Helix Motifs; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein; D050644:Succinate-Semialdehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics*; D005680:gamma-Aminobutyric Acid / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D015534:Trans-Activators; D005680:gamma-Aminobutyric Acid; D011732:Pyridoxal Phosphate; D050644:Succinate-Semialdehyde Dehydrogenase; D000612:4-Aminobutyrate Transaminase
GabR; aminotransferase; domain; pyridoxal 5′-phosphate; transcriptional regulator