Salmonella typhimurium PtsJ is a novel MocR‐like transcriptional repressor involved in regulating the vitamin B₆ salvage pathway

Abstract

The vitamin B salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B vitamers from nutrients and protein turnover to produce pyridoxal 5'-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup.

The MocR‐like transcription factors: pyridoxal 5′‐phosphate‐dependent regulators of bacterial metabolism

“…These data suggest that dimeric apo‐PtsJ binds to motifs I1 and D2 forming complex 1; in the presence of PLP, a second dimer binds to motifs I3 and D4 forming the more retarded complex 2 shown in Fig. .…”

“…With apo‐PtsJ, a low molecular weight complex (complex 1) is mainly visible, however, as the protein concentration is increased, a second more retarded complex (complex 2) appears. With holo‐PtsJ, both complexes are visible even at low protein concentration and, as protein concentration is increased, complex 1 is converted into complex 2 (as shown in EMSA analyses of Fig. , performed on purpose for this review).…”

“…So far, PLP was found to be the only B 6 vitamer able to bind at the active site of MocR‐TFs, as demonstrated by visible circular dichroism measurements . The dissociation constant ( K d ) of PLP, determined for PdxR and PtsJ by measuring the decrease of protein intrinsic fluorescence observed upon PLP binding, is around 80 n m , which is in the range observed with bacterial PLP‐dependent enzymes . GabR shows a lower affinity for PLP, with a K d determined by isothermal titration calorimetry (ITC) of about 1 μ m .…”

Transcriptional regulation of ectoine catabolism in response to multiple metabolic and environmental cues

“…Instead, a partial aminotransferase reaction occurs that initially involves the covalent binding of a system‐specific low molecular mass effector molecule to the protein‐bound PLP co‐factor (the internal aldimine) and the subsequent formation of an external aldimine between PLP and the effector molecule. These chemical reactions are transduced into a conformational change of the entire regulatory protein, which then dictates the DNA‐binding and functional properties of MocR/GabR‐type proteins to function either as activators or repressors (or both) of gene transcription (Belitsky and Sonenshein, ; Wiethaus et al ., ; Edayathumangalam et al ., ; Okuda et al ., ; Takenaka et al ., ; Park et al ., ; Tramonti et al ., ; Wu et al ., ). EnuR possesses such a covalently bound PLP molecule, and Lys‐302 present in its carboxy‐terminal aminotransferase domain has been identified through in silico modelling and mutant studies as the residue to which the co‐factor is attached (Schulz et al ., ).…”

“…MocR/GabR-type regulators are widely distributed in Gram-positive and Gram-negative bacteria but only a few of them have been functionally characterized (Belitsky and Sonenshein, 2002;Wiethaus et al, 2008;Belitsky, 2014;Okuda et al, 2015b;Takenaka et al, 2015;Tramonti et al, 2017). They possess a conserved structural organization with an N-terminal DNA-reading head containing a winged helix-turn-helix DNA-binding motif that is connected via a flexible linker region to a large carboxy-terminal effectorbinding/dimerization domain.…”