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.

Pyridoxal Kinase of Disease-causing Human Parasites: Structural and Functional Insights to Understand its Role in Drug Discovery.

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.

Thiamine-dependent regulation of mammalian brain pyridoxal kinase in vitro and in vivo.

Vitamins B1 (thiamine) and B6 (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5'-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5'-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg2+ but activating the Zn2+ -dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B1 - and B6 -dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in Kapp of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased Kapp of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness.

Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission.

The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Šresolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

Structural attributes and substrate specificity of pyridoxal kinase from Leishmania donovani.

The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5'-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5'-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of ß-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors.

Evaluation of Leishmania infantum pyridoxal kinase protein for the diagnosis of human and canine visceral leishmaniasis.

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.

Structural and functional studies on Salmonella typhimurium pyridoxal kinase: the first structural evidence for the formation of Schiff base with the substrate.

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.

Pyridoxal kinase: A vitamin B6 salvage pathway enzyme from Leishmania donovani.

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 33 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.

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

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.

Crystal structure and catalytic mechanism of pyridoxal kinase from Pseudomonas aeruginosa.

Pyridoxal kinase is a ubiquitous enzyme essential for pyridoxal 5'-phosphate (PLP) homeostasis since PLP is required for the catalytic activity of a variety of PLP-dependent enzymes involved in amino acid, lipid, and sugar metabolism as well as neurotransmitter biosynthesis. Previously, two catalytic mechanisms were proposed with regard to Pdx kinases, in which either the aspartate or the cysteine residue is involved as a catalytic residue. Because the Pdx kinase of Pseudomonas aeruginosa (PaPdxK) contains both residues, the catalytic mechanism of PaPdxK remains elusive. To elucidate the substrate-recognition and catalytic mechanisms of PaPdxK, the crystal structure of PaPdxK was determined at a 2.0 Å resolution. The PaPdxK structure possesses a channel that can accommodate substrates and a metallic cofactor. Our structure-based biochemical and mutational analyses in combination with modeling studies suggest that PaPdxK catalysis is mediated by an acid-base mechanism through the catalytic acid Asp225 and a helical dipole moment.

Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

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.

Purification, crystallization and preliminary X-ray diffraction analysis of pyridoxal kinase from Plasmodium falciparum (PfPdxK).

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=93.7 Å, ß=95°. A data set has been collected to 2 Šresolution and an initial molecular-replacement solution is described.

An inkjet-printed bioactive paper sensor that reports ATP through odour generation.

We describe an inkjet printed paper-based sensor that reports ATP by the enzyme catalysed hydrolysis of S-methyl-L-cysteine generating an odour (methyl mercaptan) that is easily detectable by the human nose.

Successful data recovery from oscillation photographs containing strong polycrystalline diffraction rings from an unknown small-molecule contaminant: preliminary structure solution of Salmonella typhimurium pyridoxal kinase (PdxK).

Pyridoxal kinase (PdxK; EC 2.7.1.35) belongs to the phosphotransferase family of enzymes and catalyzes the conversion of the three active forms of vitamin B6, pyridoxine, pyridoxal and pyridoxamine, to their phosphorylated forms and thereby plays a key role in pyridoxal 5'-phosphate salvage. In the present study, pyridoxal kinase from Salmonella typhimurium was cloned and overexpressed in Escherichia coli, purified using Ni-NTA affinity chromatography and crystallized. X-ray diffraction data were collected to 2.6 Šresolution at 100 K. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 65.11, b = 72.89, c = 107.52 Å. The data quality obtained by routine processing was poor owing to the presence of strong diffraction rings caused by a polycrystalline material of an unknown small molecule in all oscillation images. Excluding the reflections close to powder/polycrystalline rings provided data of sufficient quality for structure determination. A preliminary structure solution has been obtained by molecular replacement with the Phaser program in the CCP4 suite using E. coli pyridoxal kinase (PDB entry 2ddm) as the phasing model. Further refinement and analysis of the structure are likely to provide valuable insights into catalysis by pyridoxal kinases.

A subfamily of bacterial ribokinases utilizes a hemithioacetal for pyridoxal phosphate salvage.

Pyridoxal 5'-phosphate (PLP) is the active vitamer of vitamin B6 and acts as an essential cofactor in many aspects of amino acid and sugar metabolism. The virulence and survival of pathogenic bacteria such as Mycobacterium tuberculosis depend on PLP, and deficiencies in humans have also been associated with neurological disorders and inflammation. While PLP can be synthesized by a de novo pathway in bacteria and plants, most higher organisms rely on a salvage pathway that phosphorylates either pyridoxal (PL) or its related vitamers, pyridoxine (PN) and pyridoxamine (PM). PL kinases (PLKs) are essential for this phosphorylation step and are thus of major importance for cellular viability. We recently identified a pyridoxal kinase (SaPLK) as a target of the natural product antibiotic rugulactone (Ru) in Staphylococcus aureus. Surprisingly, Ru selectively modified SaPLK not at the active site cysteine, but on a remote cysteine residue. Based on structural and biochemical studies, we now provide insight into an unprecedented dual Cys charge relay network that is mandatory for PL phosphorylation. The key component is the reactive Cys 110 residue in the lid region that forms a hemithioactetal intermediate with the 4'-aldehyde of PL. This hemithioacetal, in concert with the catalytic Cys 214, increases the nucleophilicity of the PL 5'-OH group for the inline displacement reaction with the γ-phosphate of ATP. A closer inspection of related enzymes reveals that Cys 110 is conserved and thus serves as a characteristic mechanistic feature for a dual-function ribokinase subfamily herein termed CC-PLKs.

Turning tryptophanase into odor-generating biosensors.

An odor-based sensor system that exploits the metabolic enzyme tryptophanase (TPase) as the key component is reported. This enzyme is able to convert an odorless substrate like S-methyl-L-cysteine or L-tryptophan into the odorous products methyl mercaptan or indole. To make a biosensor, TPase was biotinylated so that it could be coupled with a molecular recognition element, such as an antibody, to develop an ELISA-like assay. This method was used for the detection of an antibody present in nM concentrations by the human nose. TPase can also be combined with the enzyme pyridoxal kinase (PKase) for use in a coupled assay to detect adenosine 5'-triphosphate (ATP). When ATP is present in the low µM concentration range, the coupled enzymatic system generates an odor that is easily detectable by the human nose. Biotinylated TPase can be combined with various biotin-labeled molecular recognition elements, thereby enabling a broad range of applications for this odor-based reporting system.

Pyridoxal 5'-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase.

Pyridoxal 5'-phosphate (PLP) is a cofactor for dozens of B(6) requiring enzymes. PLP reacts with apo-B(6) enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6) enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4'-aldehyde moiety forms covalent adducts with other compounds and non-B(6) proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6) enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.

Stoking the drug target pipeline for human African trypanosomiasis.

Trypanosoma brucei is the causative agent of African sleeping sickness, putting at risk up to 50 million people in sub-Saharan Africa. Current drug therapies are limited by toxicity and difficult treatment regimes and as the development of vaccines remains unlikely, the identification of better drugs to control this deadly disease is needed. Strategies for the identification of new lead compounds include phenotypic screening or target-based approaches. Implementation of the latter has been hampered by the lack of defined targets that are both essential and druggable. In this issue of Molecular Microbiology, Jones et al. (2012) report on the characterization of T. brucei pyridoxal kinase (PdxK), an enzyme required for the salvage of vitamin B6, an essential enzymatic cofactor. Genetic knock-down and small molecule inhibitor studies were used to demonstrate that PdxK is essential for parasite growth both in vitro and in a mouse model, providing both genetic and chemical validation of the target. An enzyme assay compatible with high-throughput screening (HTS) was developed and the X-ray crystal structure solved, showing the potential for species selective inhibition. These studies add a greatly needed additional target into the drug discovery pipeline for this deadly parasitic infection.

Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition.

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5'-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.