PdxR: a Pyridoxal 5’-Phosphate-Dependent Transcriptional Regulator Involved in the Control of PLP Biosynthesis

Discovery of unsuspected roles of vitamin B6 in many biological processes, ranging from antioxidant activity to gene expression regulation, has challenged the traditional concept of pyridoxal 5’-phosphate (PLP) as being only a cofactor involved in catalysis. Of particular interest is a subclass of understudied bacterial transcriptional regulators, named MocR, which are structurally related to PLP-dependent enzymes [1]. MocR regulators are characterized by an N-terminal domain containing a helix-turn-helix motif able to bind DNA, and a C-terminal domain called effector-binding and/or oligomerization domain, which is structurally homologous to the fold type I family of PLP-dependent enzymes (also referred to as the aspartate aminotransferase family [2]). The recent crystal structure of GabR from Bacillus subtilis, a MocR representative that regulates gamma-aminobutyric acid (GABA) metabolism, showed it to be a head-to-tail domain-swap homodimer [3]. Nonetheless, very little is known about MocR regulators, especially concerning their molecular mechanism of action and possible catalytic properties. This is a consequence of the limited number of studies so far carried out on these regulators, which have been mainly focused on transcription regulation rather than on structural and functional properties of the proteins themselves. Among all known MocR regulators, we chose PdxR for an extended biochemical characterization. This regulator is responsible for the positive transcriptional control of PLP biosynthesis through the expression of pdxST operon, coding for the two subunits of PLP synthase. In this regulatory system, PLP acts as a transcriptional repressor, thereby regulating its own synthesis [4, 5]. We carried out a screening of all available bacterial genomes in search for PdxR regulators whose gene is located adjacent to the pdxST operon and found this to be the case only for the Firmicutes phylum. Within this phylum, we focused our attention on Bacillus clausii because of its probiotic properties. We have recombinantly expressed the pdxR gene from B. clausii and purified the protein (bcPdxR) to homogeneity. Purified bcPdxR has PLP bound as a protonated Schiff base, as indicated by absorption and circular dichroism spectra. We could obtain bcPdxR in the apo-form so as to characterize the PLP binding equilibrium through CD and intrinsic fluorescence measurements. PLP is the only B6 vitamer to bind bcPdxR. Unexpectedly, ultracentrifuge analyses showed that both apo- and holo-forms of bcPdxR are in a monomeric state. DNA binding to bcPdxR was characterised through Electrophoretic Mobility Shift Assay (EMSA). Results indicated that bcPdxR binds to DNA with high affinity when in the apo-form and that PLP, but not PL or other B6 vitamers, promotes dissociation. 1. Bramucci E, et al. (2011) Biochem Biophys Res Commun. 415, 88-93. 2. Schneider G, et al. (2000) Structure. 8, R1-6. 3. Edayathumangalam R, et al. (2013) Proc Natl Acad Sci U S A. 110, 17820-5. 4. Jochmann N, et al. (2011) Microbiology. 157, 77-88. 5. Belitsky BR (2014) Mol Microbiol. 92, 1113-28.