Role of the aminotransferase domain in Bacillus subtilisGabR, a pyridoxal 5'-phosphate-dependent transcriptional regulator

MocR/GabR family proteins are widely distributed prokaryotic transcriptional regulators containing pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B6. The Bacillus subtilisGabR, probably the most extensively studied MocR/GabR family protein, consists of an N-terminal DNA-binding domain and a PLP-binding C-terminal domain that has a structure homologous to aminotransferases. GabR suppresses transcription of gabR and activates transcription of gabT and gabD, which encode gamma -aminobutyrate (G alpha beta alpha ) aminotransferase and succinate semialdehyde dehydrogenase, respectively, in the presence of PLP and GABA. In this study, we examined the mechanism underlying GabR-mediated gabTD transcription with spectroscopic, crystallographic and thermodynamic studies, focusing on the function of the aminotransferase domain. Spectroscopic studies revealed that GABA forms an external aldimine with the PLP in the aminotransferase domain. Isothermal calorimetry demonstrated that two GabR molecules bind to the 51-bp DNA fragment that contains the GabR-binding region. GABA minimally affected Delta Gbinding upon binding of GabR to the DNA fragment but greatly affected the contributions of Delta H and Delta S to Delta Gbinding. GABA forms an external aldimine with PLP and causes a conformational change in the aminotransferase domain, and this change likely rearranges GabR binding to the promoter and thus activates gabTD transcription.