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 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.

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.

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.

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.

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.

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.

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.

Chemical, genetic and structural assessment of pyridoxal kinase as a drug target in the African trypanosome.

Pyridoxal-5'-phosphate (vitamin B(6) ) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B(6) de novo and rely on uptake of B(6) vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg(-1) and a K(m)(app) with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease.

Vitamin B(6) salvage enzymes: mechanism, structure and regulation.

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

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.

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.

Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase.

Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5'-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows approximately 2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 A) shows bound ATP, PL and PLP, while D235N (2.3 A) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz.PLP.ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP.

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.

Crystal Structure of human pyridoxal kinase: structural basis of M(+) and M(2+) activation.

Pyridoxal kinase catalyzes the transfer of a phosphate group from ATP to the 5' alcohol of pyridoxine, pyridoxamine, and pyridoxal. In this work, kinetic studies were conducted to examine monovalent cation dependence of human pyridoxal kinase kinetic parameters. The results show that hPLK affinity for ATP and PL is increased manyfold in the presence of K(+) when compared to Na(+); however, the maximal activity of the Na(+) form of the enzyme is more than double the activity in the presence of K(+). Other monovalent cations, Li(+), Cs(+), and Rb(+) do not show significant activity. We have determined the crystal structure of hPLK in the unliganded form, and in complex with MgATP to 2.0 and 2.2 A resolution, respectively. Overall, the two structures show similar open conformation, and likely represent the catalytically idle state. The crystal structure of the MgATP complex also reveals Mg(2+) and Na(+) acting in tandem to anchor the ATP at the active site. Interestingly, the active site of hPLK acts as a sink to bind several molecules of MPD. The features of monovalent and divalent metal cation binding, active site structure, and vitamin B6 specificity are discussed in terms of the kinetic and structural studies, and are compared with those of the sheep and Escherichia coli enzymes.

The crystal structure of an ADP complex of Bacillus subtilis pyridoxal kinase provides evidence for the parallel emergence of enzyme activity during evolution.

Pyridoxal kinase catalyses the phosphorylation of pyridoxal, pyridoxine and pyridoxamine to their 5' phosphates and plays an important role in the pyridoxal 5' phosphate salvage pathway. The crystal structure of a dimeric pyridoxal kinase from Bacillus subtilis has been solved in complex with ADP to 2.8 A resolution. Analysis of the structure suggests that binding of the nucleotide induces the ordering of two loops, which operate independently to close a flap on the active site. Comparisons with other ribokinase superfamily members reveal that B. subtilis pyridoxal kinase is more closely related in both sequence and structure to the family of HMPP kinases than to other pyridoxal kinases, suggesting that this structure represents the first for a novel family of "HMPP kinase-like" pyridoxal kinases. Moreover this further suggests that this enzyme activity has evolved independently on multiple occasions from within the ribokinase superfamily.

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.