ABSTRACT
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.
Subject(s)
Cell Cycle Checkpoints , Gene Deletion , Leishmania donovani , Oxidation-Reduction , Pyridoxal Kinase , Leishmania donovani/genetics , Leishmania donovani/metabolism , Leishmania donovani/growth & development , Pyridoxal Kinase/metabolism , Pyridoxal Kinase/genetics , Cell Cycle Checkpoints/genetics , Animals , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Reactive Oxygen Species/metabolism , MiceABSTRACT
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.
Subject(s)
Biological Products , Hypothermia , Animals , Mice , Body Temperature Regulation , Hypothermia/chemically induced , Pyridoxal Kinase/genetics , Pyridoxine , Vitamin B 6 , Biological Products/pharmacologyABSTRACT
Cronobacter sakazakii is an opportunistic pathogen capable of causing severe infections, particularly in neonates. Despite the bacterium's strong pathogenicity, the pathogenicity of C. sakazakii is not yet well understood. Using a comparative proteomic profiling approach, we successfully identified pdxY, encoding a pyridoxal kinase involved in the recycling of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in slower growth and reduced virulence. Our study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii. The identification of pdxY as gene essential for successful pathogenesis provides a potential target for the development of new antibiotic treatments. IMPORTANCE The opportunistic pathogen Cronobacter sakazakii is known to cause severe infections, particularly in neonates, and can result in high mortality rates. In this study, we used a comparative proteomic profiling approach to identify genes essential for the successful pathogenesis of C. sakazakii. We successfully identified pdxY, encoding a pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in impaired growth and reduced virulence. This study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii, which can be a potential target for the development of new antibiotic treatments. This study highlights the importance of comparative proteomic profiling in identifying virulence factors that can be targeted for the development of new antibiotics.
Subject(s)
Cronobacter sakazakii , Cronobacter , Infant, Newborn , Humans , Vitamin B 6 , Virulence , Pyridoxal Kinase/genetics , Cronobacter sakazakii/genetics , Cronobacter sakazakii/metabolism , Proteomics , Pyridoxal Phosphate/metabolism , Pyridoxine , Anti-Bacterial Agents , Phosphates , VitaminsABSTRACT
Human parasites cause several diseased conditions with high morbidity and mortality in a large section of the population residing in various geographical areas. Nearly three billion people suffer from either one or many parasitic infections globally, with almost one million deaths annually. In spite of extensive research and advancement in the medical field, no effective vaccine is available against prominent human parasitic diseases that necessitate identification of novel targets for designing specific inhibitors. Vitamin B6 is an important ubiquitous co-enzyme that participates in several biological processes and plays an important role in scavenging ROS (reactive oxygen species) along with providing resistance to oxidative stress. Moreover, the absence of the de novo vitamin B6 biosynthetic pathway in human parasites makes this pathway indispensable for the survival of these pathogens. Pyridoxal kinase (PdxK) is a crucial enzyme for vitamin B6 salvage pathway and participates in the process of vitamers B6 phosphorylation. Since the parasites are dependent on pyridoxal kinase for their survival and infectivity to the respective hosts, it is considered a promising candidate for drug discovery. The detailed structural analysis of PdxK from disease-causing parasites has provided insights into the catalytic mechanism of this enzyme as well as significant differences from their human counterpart. Simultaneously, structure-based studies have identified small lead molecules that can be exploited for drug discovery against protozoan parasites. The present review provides structural and functional highlights of pyridoxal kinase for its implication in developing novel and potent therapeutics to combat fatal parasitic diseases.
Subject(s)
Parasites , Pyridoxal Kinase , Animals , Drug Discovery , Humans , Parasites/metabolism , Pyridoxal Kinase/chemistry , Pyridoxal Kinase/genetics , Pyridoxal Kinase/metabolism , Pyridoxine/metabolism , Vitamin B 6/chemistry , Vitamin B 6/metabolism , Vitamin B 6/pharmacologyABSTRACT
Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid act as a major neurotransmitter inhibitor in the nervous system of mammals. It also used as a precursor of bioplastics synthesis such as N-methylpyrolidone and polyamide 4. Chemical-based synthesis methods have many environmental-related issues, so efforts have been made to develop biosynthetic methods to produce GABA. Glutamate decarboxylase (GAD) transforms L-glutamate to GABA using pyridoxal 5'-phosphate (PLP) as a cofactor. Bioconversion of GABA with whole cells overexpressing the glutamate decarboxylase has advantages of fewer byproducts and rapid reaction. However, there is a bottleneck in the whole-cell bioconversion system i.e., higher GABA production require a large amount of cofactor PLP which make the process costly. Therefore, pyridoxal kinase (PdxY) able to regenerate PLP was introduced in the whole-cell system to construct a new GABA producing system. Culture and reaction conditions were optimized, and 100% conversion of 0.6 M MSG was obtained. This study reports that a competitive level of GABA production could be achieved without supplying additional PLPs.
Subject(s)
Escherichia coli , Pyridoxal Kinase , gamma-Aminobutyric Acid/biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Glutamate Decarboxylase/genetics , Pyridoxal Kinase/genetics , Pyridoxal PhosphateABSTRACT
Several approaches for efficient production of cadaverine, a bio-based diamine with broad industrial applications have been explored. Here, Serratia marcescens lysine decarboxylase (SmcadA) was expressed in E. coli; mild surfactants added in biotransformation reactions; the E. coli native lysine/cadaverine antiporter cadB, E. coli pyridoxal kinases pdxK and pdxY overexpressed and synthetic RBS libraries screened. Addition of mild surfactants and overexpression of antiporter cadB increased cadaverine biosynthesis of SmcadA. Moreover, expression of pdxY gene yielded 19.82 g/L in a reaction mixture containing added cofactor precursor pyridoxal (PL), without adding exogenous PLP. The screened synthetic RBS1, applied to fully exploit pdxY gene expression, ultimately resulted in PLP self-sufficiency, producing 27.02 g/L cadaverine using strain T7R1_PL. To boost SmcadA catalytic activity, the designed mutants Arg595Lys and Ser512Ala had significantly improved cumulative cadaverine production of 219.54 and 201.79 g/L respectively compared to the wild-type WT (181.62 g/L), after 20 h reaction. Finally, molecular dynamics simulations for WT and variants indicated that increased flexibility at the binding sites of the protein enhanced residue-ligand interactions, contributing to high cadaverine synthesis. This work demonstrates potential of harnessing different pull factors through integrated gene engineering of efficient biocatalysts and gaining insight into the mechanisms involved through MD simulations.
Subject(s)
Cadaverine/biosynthesis , Cadaverine/isolation & purification , Serratia marcescens/enzymology , Antiporters/genetics , Biotransformation/genetics , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Genetic Engineering/methods , Lysine/metabolism , Metabolic Engineering/methods , Protein Biosynthesis , Protein Processing, Post-Translational , Pyridoxal Kinase/genetics , Pyridoxal Phosphate/genetics , Serratia marcescens/metabolismABSTRACT
Visceral leishmaniasis (VL) is a highly neglected disease that is present in several countries worldwide. Present-day treatments against this disease are unsuitable, mainly due to the toxicity and/or high cost of drugs. In addition, the development of vaccines is still insufficient. In this scenario, a prompt VL diagnosis was deemed necessary, although sensitivity and/or specificity values of the tests have been. In this context, new antigenic candidates should be identified to be employed in a more precise diagnosis of canine and human VL. In this light, the present study evaluated the diagnostic efficacy of the Leishmania infantum pyridoxal kinase (PK) protein, applied in its recombinant version (rPK). In addition, one specific B-cell epitope derived of the PK sequence was predicted, synthetized, and evaluated as diagnostic marker. Results in ELISA tests showed that the antigens were highly sensitive to VL identification in dogs and human sera, presenting a low reactivity with VL-related disease samples. The recombinant A2 (rA2) protein and L. infantum antigenic preparation (SLA), used as controls, also proved to be highly sensitive in detecting symptomatic cases, although a low sensitivity was found when asymptomatic sera were analyzed. High cross-reactivity was also found when these antigens were evaluated against VL-related disease samples. The post-therapeutic serological follow-up showed that anti-rPK and anti-peptide IgG antibody levels decreased in significant levels after treatment. By contrast, the presence of high levels of the anti-rA2 and anti-SLA antibodies was still detected after therapy. In conclusion, rPK and its specific B-cell epitope should be considered for future studies as a diagnostic marker for canine and human VL.
Subject(s)
Antibodies, Protozoan/blood , Dog Diseases/diagnosis , Leishmania infantum/enzymology , Leishmaniasis, Visceral/diagnosis , Neglected Diseases/diagnosis , Protozoan Proteins/immunology , Pyridoxal Kinase/immunology , Amino Acid Sequence , Animals , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Cross Reactions , Dog Diseases/parasitology , Dogs , Enzyme-Linked Immunosorbent Assay , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Humans , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/veterinary , Neglected Diseases/parasitology , Neglected Diseases/veterinary , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Pyridoxal Kinase/chemistry , Pyridoxal Kinase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sensitivity and Specificity , Serologic TestsABSTRACT
Schistosomes are parasitic platyhelminths that currently infect >200 million people globally. The adult worms can live within the vasculature of their hosts for many years where they acquire all nutrients necessary for their survival and growth. In this work we focus on how Schistosoma mansoni parasites acquire and metabolize vitamin B6, whose active form is pyridoxal phosphate (PLP). We show here that live intravascular stage parasites (schistosomula and adult males and females) can cleave exogenous PLP to liberate pyridoxal. Of the three characterized nucleotide-metabolizing ectoenzymes expressed at the schistosome surface (SmAP, SmNPP5, and SmATPDase1), only SmAP hydrolyzes PLP. Heat-inactivated recombinant SmAP can no longer cleave PLP. Further, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to cleave PLP compared to controls. When schistosomes are incubated in murine plasma, they alter its metabolomic profile-the levels of both pyridoxal and phosphate increase over time, a finding consistent with the action of host-exposed SmAP acting on PLP. We hypothesize that SmAP-mediated dephosphorylation of PLP generates a pool of pyridoxal around the worms that can be conveniently taken in by the parasites to participate in essential, vitamin B6-driven metabolism. In addition, since host PLP-dependent enzymes play active roles in inflammatory processes, parasite-mediated cleavage of this metabolite may serve to limit parasite-damaging inflammation. In this work we also identified schistosome homologs of enzymes that are involved in intracellular vitamin B6 metabolism. These are pyridoxal kinase (SmPK) as well as pyridoxal phosphate phosphatase (SmPLP-Ph) and pyridox(am)ine 5'-phosphate oxidase (SmPNPO) and cDNAs encoding these three enzymes were cloned and sequenced. The three genes encoding these enzymes all display high relative expression in schistosomula and adult worms suggestive of robust vitamin B6 metabolism in the intravascular life stages.
Subject(s)
Alkaline Phosphatase/metabolism , Pyridoxal Phosphate/blood , Schistosoma mansoni/metabolism , Vitamin B 6/metabolism , Alkaline Phosphatase/genetics , Amino Acid Sequence , Animals , Female , Gene Expression Regulation, Developmental/genetics , Male , Mice , Phosphates/blood , Phosphoric Monoester Hydrolases/blood , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Phylogeny , Pyridoxal/blood , Pyridoxal Kinase/blood , Pyridoxal Kinase/genetics , Pyridoxal Phosphate/metabolism , Pyridoxaminephosphate Oxidase/blood , Pyridoxaminephosphate Oxidase/genetics , RNA Interference , Recombinant Proteins , Schistosoma mansoni/enzymology , Schistosoma mansoni/genetics , Schistosoma mansoni/growth & development , Sequence AlignmentABSTRACT
In eukaryotes, pyridoxal kinase (PDXK) acts in vitamin B6 salvage pathway to produce pyridoxal 5'-phosphate (PLP), the active form of the vitamin, which is implicated in numerous crucial metabolic reactions. In Drosophila, mutations in the dPdxk gene cause chromosome aberrations (CABs) and increase glucose content in larval hemolymph. Both phenotypes are rescued by the expression of the wild type human PDXK counterpart. Here we expressed, in dPdxk1 mutant flies, four PDXK human variants: three (D87H, V128I and H246Q) listed in databases, and one (A243G) found in a genetic screening in patients with diabetes. Differently from human wild type PDXK, none of the variants was able to completely rescue CABs and glucose content elicited by dPdxk1 mutation. Biochemical analysis of D87H, V128I, H246Q and A243G proteins revealed reduced catalytic activity and/or reduced affinity for PLP precursors which justify this behavior. Although these variants are rare in population and carried in heterozygous condition, our findings suggest that in certain metabolic contexts and diseases in which PLP levels are reduced, the presence of these PDXK variants could threaten genome integrity and increase cancer risk.
Subject(s)
Drosophila/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Pyridoxal Kinase/genetics , Pyridoxal Phosphate/genetics , Animals , Animals, Genetically Modified/genetics , Chromosome Aberrations , Drosophila/metabolism , Gene Expression Regulation, Enzymologic/genetics , Genomic Instability , Glucose/metabolism , Hemolymph/metabolism , Humans , Larva/genetics , Larva/metabolism , Metabolic Networks and Pathways/genetics , Mutation/genetics , Pyridoxal Kinase/metabolism , Pyridoxal Phosphate/biosynthesis , Vitamin B 6/biosynthesis , Vitamin B 6/geneticsABSTRACT
OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.
Subject(s)
Mutation/genetics , Polyneuropathies/drug therapy , Polyneuropathies/genetics , Pyridoxal Kinase/genetics , Pyridoxal Phosphate/administration & dosage , Vitamin B Complex/administration & dosage , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Dietary Supplements , Female , Gene Regulatory Networks/genetics , Humans , Male , Treatment OutcomeABSTRACT
A large number of enzymes depend on the ubiquitous cofactor pyridoxal 5' phosphate (PLP) for their activity. Pyridoxal kinase (PLK) is the key enzyme involved in the synthesis of PLP from the three forms of vitamin B6 via the salvage pathway. In the present work, we determined the unliganded structure of StPLK in a monoclinic form and its ternary complex with bound pyridoxal (PL), ADP and Mg2+ in two different tetragonal crystal forms (Form I and Form II). We found that, in the ternary complex structure of StPLK, the active site Lys233 forms a Schiff base linkage with the substrate (PL). Although formation of a Schiff base with the active site Lys229 was demonstrated in the Escherichia coli enzyme based on biochemical studies, the ternary complex of StPLK represents the first crystal structure where the Schiff bond formation has been observed. We also identified an additional site for PLP binding away from the active site in one of the ternary complexes (crystal Form I), suggesting a probable route for the product release. This is the first ternary complex structure where the modeled γ-phosphate of ATP is close enough to PL for the phosphorylation of the substrate. StPLK prefers PL over pyridoxamine as its substrate and follows a sequential mechanism of catalysis. Surface plasmon resonance studies suggest that StPLK interacts with apo-PLP-dependent enzymes with µm affinity supporting the earlier proposed direct transfer mechanism of PLP from PLK to PLP-dependent enzymes.
Subject(s)
Pyridoxal Kinase/chemistry , Pyridoxal Phosphate/chemistry , Salmonella typhimurium/enzymology , Structure-Activity Relationship , Catalysis , Catalytic Domain/genetics , Crystallography, X-Ray , Kinetics , Phosphorylation , Protein Binding/genetics , Protein Conformation , Pyridoxal Kinase/genetics , Pyridoxal Kinase/ultrastructure , Pyridoxal Phosphate/metabolism , Schiff Bases , Substrate Specificity , Vitamin B 6/chemistry , Vitamin B 6/geneticsABSTRACT
Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.
Subject(s)
Biosynthetic Pathways/genetics , Disease Resistance/genetics , Plant Diseases/genetics , Solanum lycopersicum/genetics , Vitamin B 6/biosynthesis , Antibiosis , Bacillus subtilis/physiology , Carbon-Nitrogen Lyases/genetics , Carbon-Nitrogen Lyases/metabolism , Gene Expression Regulation, Plant , Host-Pathogen Interactions , Solanum lycopersicum/metabolism , Solanum lycopersicum/microbiology , Plant Diseases/microbiology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Plant Roots/microbiology , Pyridoxal Kinase/genetics , Pyridoxal Kinase/metabolism , Xanthomonas vesicatoria/physiologyABSTRACT
Pyridoxal kinase (PdxK, EC 2.7.1.35) is an important enzyme of vitamin B6 salvage pathway which is required for phosphorylation of B6 vitamers. In the present study, pyridoxal kinase (pdxK) gene from Leishmania donovani (LdPdxK) was cloned and a 33â¯kDa protein was expressed and kinetically characterized. Site-directed mutagenesis was performed to determine the functional significance of conserved GXGD motif. Mutation of Thr229 to Ala did not affect the catalytic function of LdPdxK however Gly228, Gly230 and Asp231 were found to be indispensible for enzyme activity. To determine the role of LdPdxK in Leishmania promastigotes, LdPdxK overexpressing parasites were generated by episomal expression of the enzyme. The overexpression studies revealed the role of this enzyme in growth and infection of the parasite. In silico analysis of the human and parasite PdxK structure revealed significant differences in the active site region thus highlighting its potential as an antileishmanial drug target. Homology model of LdPdxK was built and was subjected to molecular dynamics simulations. Based on the above information, a pharmacophore was developed and shape based virtual screening was performed to identify potential and selective inhibitors against this essential enzyme. The current data suggests that LdPdxK could be a promising antileishmanial drug target.
Subject(s)
Pyridoxal Kinase/chemistry , Pyridoxal Kinase/metabolism , Amino Acid Motifs , Amino Acid Sequence , Catalytic Domain , Enzyme Activation , Gene Expression , Humans , Leishmania donovani/classification , Leishmania donovani/genetics , Leishmania donovani/metabolism , Metabolic Networks and Pathways , Models, Molecular , Mutation , Phylogeny , Protein Conformation , Pyridoxal Kinase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Vitamin B 6/chemistry , Vitamin B 6/metabolismABSTRACT
The root tip zone is regarded as the principal action site for iron (Fe) toxicity and is more sensitive than other root zones, but the mechanism underpinning this remains largely unknown. We explored the mechanism underpinning the higher sensitivity at the Arabidopsis root tip and elucidated the role of nitric oxide (NO) using NO-related mutants and pharmacological methods. Higher Fe sensitivity of the root tip is associated with reduced potassium (K+ ) retention. NO in root tips is increased significantly above levels elsewhere in the root and is involved in the arrest of primary root tip zone growth under excess Fe, at least in part related to NO-induced K+ loss via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4) and reduced root tip zone cell viability. Moreover, ethylene can antagonize excess Fe-inhibited root growth and K+ efflux, in part by the control of root tip NO levels. We conclude that excess Fe attenuates root growth by effecting an increase in root tip zone NO, and that this attenuation is related to NO-mediated alterations in K+ homeostasis, partly via SNO1/SOS4.
Subject(s)
Arabidopsis/metabolism , Iron/metabolism , Nitric Oxide/metabolism , Plant Roots/growth & development , Potassium/metabolism , Arabidopsis/drug effects , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Death , Ethylenes/metabolism , Homeostasis/drug effects , Iron/toxicity , Plant Roots/cytology , Plant Roots/drug effects , Plant Roots/metabolism , Plants, Genetically Modified , Pyridoxal Kinase/genetics , Pyridoxal Kinase/metabolism , Stress, Physiological/drug effectsABSTRACT
BACKGROUND: The identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene led us to study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of the well-studied model organism Drosophila melanogaster. The multitude of biological functions attributable to the vitamers catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk and we used RNA interference to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. We further investigated any association between this enhanced disease risk gene with the established PD model induced by expression of α-synuclein in the same neurons. We relied on the pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, to rescue the Pdxk-induced phenotypes. RESULTS: To drive the expression of Pdxk RNA interference in DA neurons of Drosophila, we used Ddc-Gal4 which drives expression in both dopaminergic and serotonergic neurons, to result in decreased longevity and compromised climbing ability, phenotypes that are strongly associated with Drosophila models of PD. The inhibition of Pdxk in the α-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when we inhibited Pdxk in the developing eye using GMR-Gal4, we found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the α-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array. CONCLUSIONS: Though Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD.
Subject(s)
Drosophila Proteins/genetics , Drosophila/genetics , Parkinson Disease/enzymology , Proto-Oncogene Proteins c-bcl-2/genetics , Pyridoxal Kinase/genetics , Animals , Disease Models, Animal , Dopa Decarboxylase/genetics , Dopa Decarboxylase/metabolism , Evolution, Molecular , Locomotion , Longevity/genetics , Neurons/metabolism , Parkinson Disease/genetics , Pyridoxal Kinase/antagonists & inhibitors , Species Specificity , Transcription Factors/genetics , Vitamin B 6/metabolism , Vitamin B Complex/metabolism , alpha-Synuclein/biosynthesisABSTRACT
Our study aims to explore the effects of lentivirus-mediated microRNA-124 (miR-124) gene-modified bone marrow mesenchymal stem cell (BMSC) transplantation on the repair of spinal cord injury (SCI) in rats. BMSCs were isolated from the bone marrow of rats. The target gene miR-124 was identified using a luciferase-reporter gene assay. Seventy-two rats were selected for construction of the SCI model, and the rats were randomly divided into the blank group, sham group, SCI group, negative control (NC) group, overexpressed miR-124 group and si-PDXK group. The mRNA expression of miR-124 and the mRNA and protein expression of pyridoxal kinase (PDXK) were detected by quantitative real-time polymerase chain reaction and western blotting. The locomotor capacity of the rats was evaluated using the Basso, Beattie and Bresnahan (BBB) scale. Brdu, neuron-specific enolase (NSE), neurofilament (NF) and microtubule-associated protein 2 (MAP2) were detected using immunohistochemistry. The expression levels of thyrotropin-releasing hormone (TRH), prostacyclin (PGI2) and gangliosides (GM) were measured using an enzyme-linked immunosorbent assay. PDXK was identified as the target gene of miR-124. The overexpressed miR-124 group exhibited higher miR-124 expression than the SCI, NC and si-PDXK groups. Compared with the SCI and NC groups, the PDXK expression was downregulated in the overexpressed miR-124 and si-PDXK groups, and the BBB scores were significantly increased 7, 21 and 35 days after transplantation. The double-labeled positive cell densities (Brdu+NSE/NF/MAP2) and the expression levels of TRH, PGI2 and GM in the overexpressed miR-124 group were significantly higher than those in the NC and SCI groups. These results indicated that miR-124 targeted PDXK to accelerate the differentiation of BMSCs into neurocytes and promote SCI repair.
Subject(s)
Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , MicroRNAs/genetics , RNAi Therapeutics , Spinal Cord Injuries/therapy , Spinal Cord Regeneration , Animals , Cells, Cultured , Epoprostenol/metabolism , Gangliosides/metabolism , Intermediate Filaments/genetics , Intermediate Filaments/metabolism , Lentivirus/genetics , MicroRNAs/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/metabolism , Pyridoxal Kinase/genetics , Pyridoxal Kinase/metabolism , Rats , Rats, Sprague-Dawley , Thyrotropin-Releasing Hormone/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Gene Expression Regulation, Bacterial , Pyridoxal Kinase/chemistry , Pyridoxaminephosphate Oxidase/chemistry , Repressor Proteins/chemistry , Salmonella typhimurium/metabolism , Vitamin B 6/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Pyridoxal Kinase/genetics , Pyridoxal Kinase/metabolism , Pyridoxal Phosphate/chemistry , Pyridoxal Phosphate/metabolism , Pyridoxaminephosphate Oxidase/genetics , Pyridoxaminephosphate Oxidase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Salmonella typhimurium/chemistry , Sequence Alignment , Structural Homology, Protein , Transcription, Genetic , Vitamin B 6/metabolismABSTRACT
Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.
Subject(s)
Bombyx/physiology , Insect Hormones/physiology , Insect Proteins/metabolism , Pyridoxal Kinase/metabolism , Pyridoxal Phosphate/biosynthesis , Pyridoxaminephosphate Oxidase/metabolism , Transcription, Genetic , Animals , Bombyx/drug effects , Bombyx/growth & development , China , Ecdysterone/antagonists & inhibitors , Ecdysterone/pharmacology , Ecdysterone/physiology , Fat Body/drug effects , Fat Body/growth & development , Fat Body/physiology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Genes, Insect/drug effects , Hormone Antagonists/pharmacology , Insect Hormones/antagonists & inhibitors , Insect Hormones/pharmacology , Insect Proteins/agonists , Insect Proteins/antagonists & inhibitors , Insect Proteins/genetics , Juvenile Hormones/pharmacology , Juvenile Hormones/physiology , Kinetics , Larva/drug effects , Larva/growth & development , Larva/physiology , Pyridoxal Kinase/antagonists & inhibitors , Pyridoxal Kinase/chemistry , Pyridoxal Kinase/genetics , Pyridoxaminephosphate Oxidase/chemistry , Pyridoxaminephosphate Oxidase/genetics , RNA, Messenger/metabolism , Salivary Glands/drug effects , Salivary Glands/growth & development , Salivary Glands/physiology , Sesquiterpenes/pharmacology , Transcription, Genetic/drug effectsABSTRACT
Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA-binding N-terminal domain and a C-terminal domain highly homologous to the pyridoxal phosphate-dependent aspartate aminotransferases. A pdxR-deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR-deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real-time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR-deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6 . Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Streptococcus mutans/genetics , Streptococcus mutans/metabolism , Transcription Factors/genetics , Vitamin B 6/metabolism , Adhesins, Bacterial/genetics , Amino Acids/metabolism , Biofilms/drug effects , Culture Media/chemistry , Gene Expression Regulation, Bacterial , Glucosyltransferases/genetics , Humans , Mutation , Operon , Pyridoxal/pharmacology , Pyridoxal Kinase/genetics , Real-Time Polymerase Chain Reaction , Streptococcus mutans/drug effects , Streptococcus mutans/growth & development , Stress, Physiological/genetics , Transaminases/genetics , Transcription Factors/metabolism , Vitamin B 6/biosynthesis , Vitamin B 6/geneticsABSTRACT
Pyridoxal kinases (PdxK) catalyze the phosphorylation of vitamin B6 precursors. Thus, these enzymes are an essential part of many metabolic processes in all organisms. The protozoan parasite Plasmodium falciparum (the main causative agent of Malaria tropica) possesses a unique de novo B6-biosynthesis pathway in addition to a interconversion pathway based on the activity of plasmodial PdxK (PfPdxK). The role of PdxK in B6 salvage has prompted previous authors to suggest PdxK as a promising target for structure-based antimalarial drug design. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of PfPdxK are reported. PfPdxK crystals have been grown in space group P21, with unit-cell parameters a=52.7, b=62.0, c=93.7â Å, ß=95°. A data set has been collected to 2â Å resolution and an initial molecular-replacement solution is described.