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Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B 6 , acts as a cofactor in many metabolic processes. In humans, PLP is produced in the reactions catalysed by pyridox(am)ine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK). Both PNPO and PDXK are involved in cancer progression of many tumours. The silencing of PNPO and PDXK encoding genes determines a strong reduction in tumour size and neoplastic cell invasiveness in models of acute myeloid leukaemia (in the case of PDXK) and ovarian and breast cancer (in the case of PNPO). In the present work, we demonstrate that pyridoxilidenerhodanine 5'-phosphate (PLP-R), a PLP analogue that has been tested by other authors on malignant cell lines reporting a reduction in proliferation, inhibits PNPO in vitro following a mixed competitive and allosteric mechanism. We also show that the unphosphorylated precursor of this inhibitor (PL-R), which has more favourable pharmacokinetic properties according to our predictions, is phosphorylated by PDXK and therefore transformed into PLP-R. On this ground, we propose the prototype of a novel prodrug-drug system as a useful starting point for the development of new, potential, antineoplastic agents., (© 2024 Federation of European Biochemical Societies.)

Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B 6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA metabolism. The concerted action of PLP-dependent serine hydroxymethyltransferase (SHMT) and thymidylate synthase (TS) leads to the biosynthesis of thymidylate (dTMP), which plays an essential function in DNA synthesis and repair. PLP deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, rising the hypothesis that an altered 1C metabolism may be involved. To test this hypothesis, we used Drosophila as a model system and found, firstly, that in PLP deficient larvae SHMT activity is reduced by 40%. Second, we found that RNAi-induced SHMT depletion causes chromosome damage rescued by PLP supplementation and strongly exacerbated by PLP depletion. RNAi-induced TS depletion causes severe chromosome damage, but this is only slightly enhanced by PLP depletion. dTMP supplementation rescues CABs in both PLP-deficient and PLP-proficient SHMT RNAi . Altogether these data suggest that a reduction of SHMT activity caused by PLP deficiency contributes to chromosome damage by reducing dTMP biosynthesis. In addition, our work brings to light a gene-nutrient interaction between SHMT decreased activity and PLP deficiency impacting on genome stability that may be translated to humans., (© 2023 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3K RNAi flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3K RNAi larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications., (© 2022 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Several variants of the enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), responsible for a rare form of vitamin B 6 -dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5'-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5'-phosphate product.

Vitamin B 6 is an ensemble of six interconvertible vitamers: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5'-phosphate derivatives, PNP, PMP, and PLP. Pyridoxal 5'-phosphate is a coenzyme in a variety of enzyme reactions concerning transformations of amino and amino acid compounds. This review summarizes all known and putative PLP-binding proteins found in the Escherichia coli MG1655 proteome. PLP can have toxic effects since it contains a very reactive aldehyde group at its 4' position that easily forms aldimines with primary and secondary amines and reacts with thiols. Most PLP is bound either to the enzymes that use it as a cofactor or to PLP carrier proteins, protected from the cellular environment but at the same time readily transferable to PLP-dependent apoenzymes. E. coli and its relatives synthesize PLP through the seven-step deoxyxylulose-5-phosphate (DXP)-dependent pathway. Other bacteria synthesize PLP in a single step, through a so-called DXP-independent pathway. Although the DXP-dependent pathway was the first to be revealed, the discovery of the widespread DXP-independent pathway determined a decline of interest in E. coli vitamin B 6 metabolism. In E. coli , as in most organisms, PLP can also be obtained from PL, PN, and PM, imported from the environment or recycled from protein turnover, via a salvage pathway. Our review deals with all aspects of vitamin B 6 metabolism in E. coli , from transcriptional to posttranslational regulation. A critical interpretation of results is presented, in particular, concerning the most obscure aspects of PLP homeostasis and delivery to PLP-dependent enzymes.

A perturbed uptake of micronutrients, such as minerals and vitamins, impacts on different human diseases, including cancer and neurological disorders. Several data converge towards a crucial role played by many micronutrients in genome integrity maintenance and in the establishment of a correct DNA methylation pattern. Failure in the proper accomplishment of these processes accelerates senescence and increases the risk of developing cancer, by promoting the formation of chromosome aberrations and deregulating the expression of oncogenes. Here, the main recent evidence regarding the impact of some B vitamins on DNA damage and cancer is summarized, providing an integrated and updated analysis, mainly centred on vitamin B 6 . In many cases, it is difficult to finely predict the optimal vitamin rate that is able to protect against DNA damage, as this can be influenced by a given individual's genotype. For this purpose, a precious resort is represented by model organisms which allow limitations imposed by more complex systems to be overcome. In this review, we show that Drosophila can be a useful model to deeply understand mechanisms underlying the relationship between vitamin B 6 and genome integrity.

The vitamin B 6 salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B 6 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 6 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 6 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., (© 2016 Federation of European Biochemical Societies.)

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., (© 2015 FEBS.)

Vitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-enzymes in a "salvage pathway", which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5'-phosphate oxidase. Once it is made, pyridoxal 5'-phosphate is targeted to the dozens of different apo-B(6) enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5'-phosphate to the apo-B(6)-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5'-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5'-phosphate formation. Deficiency of pyridoxal 5'-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5'-phosphate deficiency. Understanding the exact role of vitamin B(6) in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B(6)-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology., (Copyright © 2010 Elsevier B.V. All rights reserved.)