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.