Pyridoxal Kinase Research Articles

B-016 Evaluation of pyridoxamine phosphate (PAMP) for AST/ALT reagents with recombinant human apoenzymes

Abstract Background ApoAST/ALT, which have lost a coenzyme such as pyridoxal phosphate (PALP), are normally present at low levels in serum. To form the holoenzyme, IFCC recommends adding PALP into AST/ALT reagents. However, commercial reagents often do not use PALP due to its instability. In recent years, pyridoxamine phosphate (PAMP), a more stable coenzyme, has been marketed. However, the differences in holo formation ability and reagent stability between their coenzymes are not well understood. In this study, we focused on the preparation of PAMP and recombinant human ApoAST/ALT (rhApoAST/ALT), followed by fundamental investigations into their use as AST/ALT reagents. Methods PAMP was synthesized from pyridoxamine through an enzymatic reaction using pyridoxal kinase. The reaction product was purified to achieve a purity of >99% in HPLC. rhApoAST/ALT were expressed in Escherichia coli and purified with chromatography. AST/ALT assay was performed on a Hitachi 7180 automatic analyzer. PALP reagents for AST/ALT were designed to correspond with the final concentration of the IFCC method. PAMP reagents were prepared with PAMP instead of PALP. To evaluate the holo formation ability of PALP and PAMP reagents, we measured a dilution series of rhApoAST/ALT. To assess stability, the reagents were stored at 11°C for 3 months and then measured same. The reagents were subjected to content analysis by HPLC. Results Similar results were shown between coenzymes in the evaluation of holo formation ability, and high correlations were observed. As for the stability test, the values of PALP reagents decreased after 3 months, while the values of PAMP reagents did not. Content analysis showed that a spontaneous transamination occurred in PALP reagents. These results were observed at both AST and ALT reagents. Conclusions In AST/ALT reagents, both PAMP and PALP can activate apoenzymes. While PAMP is stable, PALP causes a decrease in reagent performance due to a spontaneous transamination.

The Z isomer of pyridoxilidenerhodanine 5'-phosphate is an efficient inhibitor of human pyridoxine 5'-phosphate oxidase, a crucial enzyme in vitamin B6 salvage pathway and a potential chemotherapeutic target.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, 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.

Highly efficient bio-production of putrescine from L-arginine with arginase and L-ornithine decarboxylase in engineered Escherichia coli

To achieve industrial-scale putrescine production, a high efficient bio-synthesis of putrescine involving arginase (ARG, EC 3.5.3.1) and L-ornithine decarboxylase was evaluated here. Among the four arginases tested, ARGBT from Bos Taurus showed the highest activity (1966 U/mg). Compared to the L-arginine decarboxylase (ADC) pathway, the strain expressing ARGBT and L-ornithine decarboxylase (SpeC) produced 28.7 g/L putrescine, a 38.6 % increase. Two pyridoxal phosphate (PLP) salvage pathways were evaluated, and the strain BL-PTac-PdxK co-expressed pyridoxal kinase (PdxK) performed better. D-Glucose was used as the co-substrate to improve the putrescine titer further. Under optimal conditions, 43.6 g/L putrescine was produced from 87.1 g/L L-arginine, and 76 g/L putrescine was synthesized on a 0.5 L scale. Using L-arginine fermentation broth (60 g/L) as the substrate, a titer of 30 g/L putrescine was achieved. This efficient biotransformation process presented here enables feasible industrial-scale putrescine production.

Exploring the role of pyridoxal kinase: a key player in vitamin B6 metabolism

Exploring the role of pyridoxal kinase: a key player in vitamin B6 metabolism

Butyrate inhibits the malignant biological behaviors of breast cancer cells by facilitating cuproptosis-associated gene expression

BackgroundButyrate is a common short-chain fatty acids (SCFA), and it has been demonstrated to regulate the development of breast cancer (BC), while the underlying mechanism is still unreported.MethodsGas chromatography was used to measure the amounts of SCFA (acetate, propionate, and butyrate) in the feces. Cell viability was measured by the CCK-8 assay. The wound healing assay demonstrated cell migration, and the transwell assay demonstrated cell invasion. The levels of protein and gene were determined by western blot assay and RT-qPCR assay, respectively.ResultsThe levels of SCFA were lower in the faecal samples from BC patients compared to control samples. In cellular experiments, butyrate significantly suppressed the cell viability, migration and invasion of T47D in a dose-dependent manner. In animal experiments, butyrate effectively impeded the growth of BC tumors. Toll like receptor 4 (TLR4) was highly expressed in the tumors from BC patients. Butyrate inhibited the expression of TLR4. In addition, butyrate promoted the expression of cuproptosis-related genes including PDXK (pyridoxal kinase) and SLC25A28 (solute carrier family 25 member 28), which was lowly expressed in BC tumors. Importantly, overexpression of TLR4 can reverses the promotion of butyrate to PDXK and SLC25A28 expression and the prevention of butyrate to the malignant biological behaviors of T47D cells.ConclusionIn summary, butyrate inhibits the development of BC by facilitating the expression of PDXK and SLC25A28 through inhibition of TLR4. Our investigation first identified a connection among butyrate, TLR4 and cuproptosis-related genes in BC progression. These findings may provide novel target for the treatment of BC.

Identification of testis development-related genes by combining Iso-Seq and RNA-Seq in Zeugodacus tau.

Introduction: Zeugodacus tau (Walker) is an invasive pest. An effective method to control this pest is the sterile insect technique (SIT). To better apply this technique, it is necessary to understand testis development progression. Methods: Differentially expressed genes (DEGs) during testis development were analyzed by PacBio Iso-Seq and RNA-seq. Results: RNA-Seq library of Z. tau testes on day 1, 6, and 11 post eclosion were constructed. We identified 755 and 865 differentially expressed genes in the comparisons of T6 (testes on day 6) vs. T1 and T11 vs. T1, respectively. The KEGG pathway analysis showed that the DEGs were significantly enriched in retinol metabolism, vitamin B6 metabolism, and ascorbate and aldarate metabolism pathways. Knockdown of retinol dehydrogenase 12-like (rdh12-like), pyridoxal kinase (pdxk) and regucalcin (rgn), the representative gene in each of the above 3 pathways, reduced the hatching rate of Z. tau offspring. In addition, we identified 107 Drosophila spermatogenesis-related orthologous genes in Z. tau, of which innexin 2 (inx2) exhibited significantly up-regulated expression throughout testis development, and the knockdown of this gene reduced offspring hatching rate. Discussion: Our data indicated that rdh12-like, pdxk, rgn, and inx2 genes were related to testis development, and they were conserved in tephritid species. These results suggested that this gene might have the same function in tephritid. The findings provide an insight into testis development and spermatogenesis in tephritid species.

Pyridoxal 5'-Phosphate Biosynthesis by Pyridox-(am)-ine 5'-Phosphate Oxidase: Species-Specific Features.

Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.

Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani

Pyridoxal kinase (PdxK) is a vitamin B6 salvage pathway enzyme which produces pyridoxal phosphate. We have investigated the impact of PdxK deletion in Leishmania donovani on parasite survivability, infectivity and cellular metabolism. LdPdxK mutants were generated by gene replacement strategy. All mutants showed significant reduction in growth in comparison to wild type. For PdxK mediated biochemical perturbations, only heterozygous mutants and complementation mutants were used as the growth of null mutants were compromised. Heterozygous mutant showed reduction invitro infectivity and higher cytosolic and mitochondrial ROS levels. Glutathione levels decreased significantly in heterozygous mutant indicating its involvement in cellular oxidative metabolism. Pyridoxal kinase gene deletion resulted in reduced ATP levels in parasites and arrest at G0/G1 phase of cell cycle. All these perturbations were rescued by PdxK gene complementation. This is the first report to confirm that LdPdxK plays an indispensable role in cell survival, pathogenicity, redox metabolism and cell cycle progression of L. donovani parasites. These results provide substantial evidence supporting PdxK as a therapeutic target for the development of specific antileishmanial drug candidates.

Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate.

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 μM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

Discovery and characterization of natural product luteolin as an effective inhibitor of human pyridoxal kinase

Pyridoxal kinase (PDXK) is an essential enzyme in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B6, which plays a pivotal role in maintaining the enzyme activity necessary for cell metabolism. Thus, PDXK has garnered attention as a potential target for metabolism regulation and tumor therapy. Despite this interest, existing PDXK inhibitors have faced limitations, including weak suppressive activity, unclear mechanisms of action, and associated toxic side effects. In this study, we present the discovery of a novel PDXK inhibitor, luteolin, through a high-throughput screening approach based on enzyme activity. Luteolin, a natural product, exhibits micromolar-level affinity for PDXK and effectively inhibits the enzyme's activity in vitro. Our crystal structures reveal that luteolin occupies the ATP binding pocket through hydrophobic interactions and a weak hydrogen bonding pattern, displaying reversible characteristics as confirmed by biochemical assays. Moreover, luteolin disrupts vitamin B6 metabolism by targeting PDXK, thereby inhibiting the proliferation of leukemia cells. This research introduces a novel screening method for identifying high-affinity and potent PDXK inhibitors and sheds light on clarification of the structural mechanism of PDXK-luteolin for subsequent structure optimization of inhibitors.

Protein-protein interfaces as druggable targets: a common motif of the pyridoxal-5′-phosphate-dependent enzymes to receive the coenzyme from its producer

Pyridoxal-5′-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP may modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in a multitude of the PLP-dependent enzymes for binding the limited number of the PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5′-phosphate oxidase (PNPO) and the PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the PLP acceptors of folds I and II, while PLPBP - with those of folds III and V. Aligning the sequences and 3D structures of the identified interactors of PdxK and PNPO, we have found a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand and residual loops. Pathogenicity of mutations in human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of the coenzyme binding. The enzyme-specific amino acid residues of the common motif may be used to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

New Insights Into Pyridoxal Kinase Inhibitors and Their Antileukemic Effects.

Pyridoxal kinase (PDXK) plays a pivotal role as an essential enzyme in cellular processes. It catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine to generate pyridoxal 5'-phosphate (PLP), the bioactive form of vitamin B6. An intriguing link has emerged between elevated expression levels of PDXK and PLP and various types of carcinomas, including leukemia. Leukemic cells have an increased need for vitamin B6 to sustain their survival and rapid growth, highlighting the potential of targeting PDXK-PLP as a promising therapeutic target in cancer treatment. To discover a novel and promising PDXK inhibitor, we conducted a comprehensive screening of compounds derived from both natural sources and drug-like databases. Our approach involved employing structure-based virtual screening and molecular docking techniques to attenuate the phosphorylation of PLP. Among the top six compounds, ZINC095099376 (referred to as C03) emerged as the most potent inhibitor of PDXK, primarily due to its exceptional binding affinity and remarkable specificity for the target protein. Furthermore, our investigation revealed that compound C03 establishes crucial interactions with key residues within the substrate binding site, indicating that it binds at the same site as the co-crystallized ligand. Remarkably, compound C03 inhibited the endogenous PDXK expression, showed anti-proliferative activity, and triggered an intrinsic pathway for apoptosis via the activation of key apoptotic factors in leukemic cells. In summary, these findings strongly indicate that compound C03 holds promise as a novel inhibitor of PDXK, offering the potential for the development of effective treatments for leukemia.

Cheminformatics-based discovery of new organoselenium compounds with potential for the treatment of cutaneous and visceral leishmaniasis

Leishmaniasis affects more than 12 million humans globally and a further 1 billion people are at risk in leishmaniasis endemic areas. The lack of a vaccine for leishmaniasis coupled with the limitations of existing anti-leishmanial therapies prompted this study. Cheminformatic techniques are widely used in screening large libraries of compounds, studying protein-ligand interactions, analysing pharmacokinetic properties, and designing new drug molecules with great speed, accuracy, and precision. This study was undertaken to evaluate the anti-leishmanial potential of some organoselenium compounds by quantitative structure-activity relationship (QSAR) modeling, molecular docking, pharmacokinetic analysis, and molecular dynamic (MD) simulation. The built QSAR model was validated (R2 train = 0.8646, R2 test = 0.8864, Q2 = 0.5773) and the predicted inhibitory activity (pIC50) values of the newly designed compounds were higher than that of the template (Compound 6). The new analogues (6a, 6b, and 6c) showed good binding interactions with the target protein (Pyridoxal kinase, PdxK) while also presenting excellent drug-likeness and pharmacokinetic profiles. The results of density functional theory, MD simulation, and molecular mechanics generalized Born surface area (MM/GBSA) analyses suggest the favourability and stability of protein-ligand interactions of the new analogues with PdxK, comparing favourably well with the reference drug (Pentamidine). Conclusively, the newly designed compounds could be synthesized and tested experimentally as potential anti-leishmanial drug molecules. Communicated by Ramaswamy H. Sarma

Metabolic modeling predicts unique drug targets in Borrelia burgdorferi.

Lyme disease is often treated using long courses of antibiotics, which can cause side effects for patients and risks the evolution of antimicrobial resistance. Narrow-spectrum antimicrobials would reduce these risks, but their development has been slow because the Lyme disease bacterium, Borrelia burgdorferi, is difficult to work with in the laboratory. To accelerate the drug discovery pipeline, we developed a computational model of B. burgdorferi's metabolism and used it to predict essential enzymatic reactions whose inhibition prevented growth in silico. These predictions were validated using small-molecule enzyme inhibitors, several of which were shown to have specific activity against B. burgdorferi. Although the specific compounds used are not suitable for clinical use, we aim to use them as lead compounds to develop optimized drugs targeting the pathways discovered here.

Natural product P57 induces hypothermia through targeting pyridoxal kinase

Induction of hypothermia during hibernation/torpor enables certain mammals to survive under extreme environmental conditions. However, pharmacological induction of hypothermia in most mammals remains a huge challenge. Here we show that a natural product P57 promptly induces hypothermia and decreases energy expenditure in mice. Mechanistically, P57 inhibits the kinase activity of pyridoxal kinase (PDXK), a key metabolic enzyme of vitamin B6 catalyzing phosphorylation of pyridoxal (PL), resulting in the accumulation of PL in hypothalamus to cause hypothermia. The hypothermia induced by P57 is significantly blunted in the mice with knockout of PDXK in the preoptic area (POA) of hypothalamus. We further found that P57 and PL have consistent effects on gene expression regulation in hypothalamus, and they may activate medial preoptic area (MPA) neurons in POA to induce hypothermia. Taken together, our findings demonstrate that P57 has a potential application in therapeutic hypothermia through regulation of vitamin B6 metabolism and PDXK serves as a previously unknown target of P57 in thermoregulation. In addition, P57 may serve as a chemical probe for exploring the neuron circuitry related to hypothermia state in mice.

Vitamin B6 is governed by the local compartmentalization of metabolic enzymes during growth.

Vitamin B6 is a vital micronutrient across cell types and tissues, and dysregulated B6 levels contribute to human disease. Despite its importance, how B6 vitamer levels are regulated is not well understood. Here, we provide evidence that B6 dynamics are rapidly tuned by precise compartmentation of pyridoxal kinase (PDXK), the rate-limiting B6 enzyme. We show that canonical Wnt rapidly led to the accumulation of inactive B6 by shunting cytosolic PDXK into lysosomes. PDXK was modified with methyl-arginine Degron (MrDegron), a protein tag for lysosomes, which enabled delivery via microautophagy. Hyperactive lysosomes resulted in the continuous degradation of PDXK and B6 deficiency that promoted proliferation in Wnt-driven colorectal cancer (CRC) cells. Pharmacological or genetic disruption of the coordinated MrDegron proteolytic pathway was sufficient to reduce CRC survival in cells and organoid models. In sum, this work contributes to the repertoire of micronutrient-regulated processes that enable cancer cell growth and provides insight into the functional impact of B6 deficiencies for survival.

Molecular scaffold recognition of drug molecules against essential genes of Leishmania donovani using biocomputing approach

Leishmania donovani, an obligatory intracellular flagellate pathogen, is the underlying cause of visceral leishmaniasis (VL), a fatal disease that poses a significant challenge to existing therapeutic approaches and leads to human mortality. In an endeavor to find an antileishmanial drug to combat VL, we aimed to assess the approved drug molecules against the specific drug targets of VL. In this study, a theoretical method was used to select two essential therapeutic targets (pyridoxal kinase [PK] and sterol alpha-14 demethylase [SDM]) which were present in both the data set of essential genes and drug target proteins. The selected PK and SDM proteins in L. donovani play pivotal roles as essential enzymes in the crucial vitamin B6 salvage and sterol biosynthesis pathways, respectively, leading to pathogenicity in humans. In addition to that drugs were gathered from the DrugBank and Drug Central databases and 325 (out of 4867) compounds having anti-parasitic properties were screened by PASS analysis. Consequently, three ligands (referred to as Lig_1, Lig_2, and Lig_3) were chosen based on their elevated Pa values, docking scores, and notable medicinal applications. Moreover, the result obtained from MD simulation suggests Lig_1 [Nitazoxanide (PubChem ID-41684)] does not affect the structural integrity of both targets. Additionally, evaluation of total binding energies by MMPBSA analysis showed stronger binding of Lig_1 with PK and SDM is -100.71 and -175.61 kJ/mol, respectively compared to others. As a whole, the methodology employed in this research involves the simultaneous identification of suitable protein targets and potential inhibitors. Through this investigation, we have demonstrated that compounds derived from a biocomputing approach exhibit interaction mechanisms as inhibitors against drug targets, offering a promising avenue for addressing VL.

Identification of high-affinity pyridoxal kinase inhibitors targeting cancer therapy: an integrated docking and molecular dynamics simulation approach

Pyridoxal kinase (PDXK) is a vitamin B6-dependent transferase enzyme encoded by the PDXK gene, crucial for leukemic cell proliferation. Disruption of its activity causes altered metabolism and reduced levels of nucleotides and polyamines. PDXK and pyridoxal 5'-phosphate (PLP) are overexpressed in various carcinomas, making them promising targets for drug design against cancer. Targeting PDXK may hold promise as a therapeutic approach for cancer treatment. This study focused on discovering potential inhibitors that could selectively interrupt the binding of pyridoxal phosphate (PLP) to pyridoxal kinase (PDXK). A commercially available library of 7,28,747 natural and druglike compounds was virtually screened using a molecular docking approach to target the substrate binding pocket of PDXK. Six promising inhibitors were identified, and all-atom molecular dynamics simulations were conducted on the PDXK-ligand complexes for 100 ns to assess their binding conformational stability. The simulation results indicated that the binding of ZINC095099376, ZINC01612996, ZINC049841390, ZINC095098959, ZINC01482077, and ZINC03830976 induced a slight structural change and stabilized the PDXK structure. This analysis provided valuable information about the critical residues involved in the PDXK-PLP complex formation and can be utilized in designing specific and effective PDXK inhibitors. According to this study, these compounds could be developed as anticancer agents targeting PDXK as a potential candidate for further study. Communicated by Ramaswamy H. Sarma

Protein–Protein Interfaces as Druggable Targets: A Common Motif of the Pyridoxal-5′-Phosphate-Dependent Enzymes to Receive the Coenzyme from Its Producers

Pyridoxal-5′-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP can modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in the multitude of PLP-dependent enzymes for binding the limited number of PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5′-phosphate oxidase (PNPO), and PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the most abundant PLP acceptors belonging to structural folds I and II, while PLPBP – with those belonging to folds III and V. Aligning sequences and 3D structures of the identified interactors of PdxK and PNPO, we have identified a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand, and residual loops. Pathogenicity of mutations in the human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of coenzyme binding. The enzyme-specific amino acid residues of the common motif may be useful to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

Vitamin B6 regulates IL-33 homeostasis to alleviate type 2 inflammation.

Interleukin-33 (IL-33) is a crucial nuclear cytokine that induces the type 2 immune response and maintains immune homeostasis. The fine-tuned regulation of IL-33 in tissue cells is critical to control of the type 2 immune response in airway inflammation, but the mechanism is still unclear. Here, we found that healthy individuals had higher phosphate-pyridoxal (PLP, an active form of vitamin B6) concentrations in the serum than asthma patients. Lower serum PLP concentrations in asthma patients were strongly associated with worse lung function and inflammation. In a mouse model of lung inflammation, we revealed that PLP alleviated the type 2 immune response and that this inhibitory effect relied on the activity of IL-33. A mechanistic study showed that in vivo, pyridoxal (PL) needed to be converted into PLP, which inhibited the type 2 response by regulating IL-33 stability. In mice heterozygous for pyridoxal kinase (PDXK), the conversion of PL to PLP was limited, and IL-33 levels were increased in the lungs, aggravating type 2 inflammation. Furthermore, we found that the mouse double minute 2 homolog (MDM2) protein, an E3 ubiquitin-protein ligase, could ubiquitinate the N-terminus of IL-33 and sustain IL-33 stability in epithelial cells. PLP reduced MDM2-mediated IL-33 polyubiquitination and decreased the level of IL-33 through the proteasome pathway. In addition, inhalation of PLP alleviated asthma-related effects in mouse models. In summary, our data indicate that vitamin B6 regulates MDM2-mediated IL-33 stability to constrain the type 2 response, which might help develop a potential preventive and therapeutic agent for allergy-related diseases.