title: Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii.
abstract: Pyridoxal 5'-phosphate (PLP), the well-known active form of vitamin B6 , is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N-terminal helix-turn-helix motif that binds DNA and an effector-binding C-terminal domain homologous to PLP-dependent enzymes. Although it is known that PLP acts as an anti-activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA-binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B6 vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA-binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.
authors.affiliation: Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Roma, Italy.|Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Tramonti
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Fiascarelli
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Milano
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Martino L
authors.initials: ML
authors.lastname: di Salvo
authors.affiliation: Istituto di Biologia Ambientale e Forestale, Consiglio Nazionale delle Ricerche, Monterotondo Scalo, Roma, Italy.
authors.forename: Isabel
authors.initials: I
authors.lastname: Nogués
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Pascarella
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Contestabile
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D019936:Probiotics; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D025101:Vitamin B 6 / Q000096:biosynthesis*
chemical_list: D000596:Amino Acids; D015534:Trans-Activators; D025101:Vitamin B 6; D004247:DNA
keywords: Bacillus clausii; MocR; PdxR; pyridoxal 5′-phosphate biosynthesis; transcriptional regulation