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

• Published in: The FEBS journal 2017-02, Vol.284 (3), p.466-484
• Main Authors: Tramonti, Angela, Milano, Teresa, Nardella, Caterina, Salvo, Martino L., Pascarella, Stefano, Contestabile, Roberto
• Format: Article
• Language: English
• Subjects: Binding sites; Deoxyribonucleic acid; DNA; Gene expression; Gene regulation; Gene sequencing; Homeostasis; Kinases; MocR; Nucleotide sequence; Nutrients; Phosphates; Polo-like kinase 1; Protein turnover; Proteins; PtsJ; Pyridoxal kinase; Pyridoxine; Salmonella; Salmonella Typhimurium; Salvage; Stoichiometry; Subgroups; Vitamin B; Vitamin B6; vitamin B6 salvage pathway
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.13994

The vitamin B6 salvage pathway, involving pyridoxine 5′‐phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B6 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 B6 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 B6 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. PtsJ from Salmonella typhimurium, a novel MocR‐like transcriptional regulator, represses expression of the pdxK gene, encoding vitamin B6 salvage pathway enzyme pyridoxal kinase. PtsJ binds to four repeated nucleotide sequences on the ptsJ‐pdxK intergenic region. Binding to DNA is promoted by pyridoxal phosphate (PLP) that shifts binding stoichiometry from a 2 : 1 to a 4 : 1 protein subunits/DNA molar ratio.