title: PLP and GABA trigger GabR-mediated transcription regulation inBacillus subtilisvia external aldimine formation.
abstract: TheBacillus subtilisprotein regulator of thegabTDoperon and its own gene (GabR) is a transcriptional activator that regulates transcription of γ-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic γ-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) fromEscherichia colias a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.
authors.affiliation: Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL 60660.
authors.forename: Rui
authors.initials: R
authors.lastname: Wu
authors.affiliation: GM/CA@APS, The X-Ray Science Division, the Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Sanishvili
authors.affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111.
authors.forename: Boris R
authors.initials: BR
authors.lastname: Belitsky
authors.affiliation: Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113.|Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113.
authors.forename: Jose I
authors.initials: JI
authors.lastname: Juncosa
authors.affiliation: Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113.|Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113.
authors.forename: Hoang V
authors.initials: HV
authors.lastname: Le
authors.affiliation: Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454.
authors.forename: Helaina J S
authors.initials: HJ
authors.lastname: Lehrer
authors.affiliation: Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL 60660.
authors.forename: Michael
authors.initials: M
authors.lastname: Farley
authors.affiliation: Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113.|Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Silverman
authors.affiliation: Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454; dliu@luc.edu gpetsko@med.cornell.edu.|Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY 10065.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Petsko
authors.affiliation: Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Ringe
authors.affiliation: Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL 60660; dliu@luc.edu gpetsko@med.cornell.edu.
authors.forename: Dali
authors.initials: D
authors.lastname: Liu
mesh_terms: D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015964:Gene Expression Regulation, Bacterial; D009682:Magnetic Resonance Spectroscopy; D009876:Operon; D010421:Pentanoic Acids / Q000378:metabolism / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D000072417:Protein Domains; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012545:Schiff Bases; D014158:Transcription, Genetic; D005680:gamma-Aminobutyric Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010421:Pentanoic Acids; D012545:Schiff Bases; D005680:gamma-Aminobutyric Acid; D011732:Pyridoxal Phosphate; C032606:4-amino-5-fluoropentanoic acid
keywords: GabR; MocR; PLP; external aldimine; transcription activation