The goals of the present project are designed to investigate the molecular mechanism of regulation of the pathway of glutamate generation from gamma-aminobutyrate (GABA) in Bacillus subtilis. The most interesting component of this pathway is GabR, a pyridoxal 5'-phosphate (PLP)- and GABA-dependent transcriptional activator that regulates expression of an operon that encodes two enzymes of GABA utilization. GabR is a member of a novel family of unusual proteins that apparently evolved by joining a DNA-binding domain to an aminotransferase domain. Of particular interest is the requirement for PLP (an essential catalytic cofactor of aminotransferases) and the apparent PLP-binding site of GabR for transcriptional activation. The preliminary data indicate that, in order to activate expression of the target genes, GabR performs (at least) a partial aminotransferase reaction. The main goal of the project will be to show that GabR activity as a transcriptional activator is modulated by GabR-catalyzed transamination of its cofactor PLP, using GABA as an amino group donor. This goal will be achieved by analyzing interactions between GabR and its coactivators, by utilizing structural analogs of PLP and GABA, and by characterization of mutant GabR proteins defective in transcription activation or able to activate transcription constitutively. GabR properties as a specific DNA-binding protein will be characterized. GabR will be the first member of this protein family to be subjected to detailed biochemical and molecular analysis and will provide clues to the mechanism of action of other GabR-like proteins. Because the aminotransferase reaction requires an amino group-containing substrate, the prediction is that all GabR-like proteins have such compounds as their effectors.