Human Pyridoxal Kinase Research Articles

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.

An Evolutionary Marker of the Ribokinase Superfamily Is Responsible for Zinc-Mediated Regulation of Human Pyridoxal Kinase

The ribokinase superfamily catalyzes the phosphorylation of a vast diversity of substrates, and its members are characterized by the conservation of a common structural fold along with highly conserved sequence motifs responsible for phosphoryl transfer (GXGD) and stabilization of the metal-nucleotide complex (NXXE). Recently, a third motif (HXE) exclusive from ADP-dependent enzymes was identified, with its glutamic acid participating in water-mediated interactions with the metal-nucleotide complex and in stabilization of the ternary complex during catalysis. In this work, we bioinformatically determine that the aspartic acid of another motif (DPV), exclusively found in hydroxyethyl thiazole (THZK), hydroxymethyl pyrimidine (HMPK) and pyridoxal kinases (PLK), is structurally equivalent to the acidic residue in the HXE motif. Moreover, this residue is highly conserved among all ribokinase superfamily members. To determine whether the functional role of the DPV motif is similar to the HXE motif, we employed molecular dynamics simulations using crystal structures of phosphoryl donor substrate-complexed THZK and PLK, showing that its aspartic acid participated in water-mediated or direct interactions with the divalent metal of the metal-nucleotide complex. Lastly, enzyme kinetic assays on human PLK, an enzyme that utilizes zinc, showed that site-directed mutagenesis of the aspartic acid from the DPV motif abolishes the inhibition of this enzyme by increasing free zinc concentrations. Altogether, our results highlight that the DPV and HXE motifs are evolutionary markers of the functional and structural divergence of the ribokinase superfamily and evidence the role of the DPV motif in the interaction with both free and nucleotide-complexed divalent metals in the binding site of these enzymes.

The expression of four pyridoxal kinase (PDXK) human variants in Drosophila impacts on genome integrity

In eukaryotes, pyridoxal kinase (PDXK) acts in vitamin B6salvage 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.

Customizing Functionalized Cofactor Mimics to Study the Human Pyridoxal 5′-Phosphate-Binding Proteome

SummaryPyridoxal 5′-phosphate (PLP) is a versatile cofactor that catalyzes a plethora of chemical transformations within a cell. Although many human PLP-dependent enzymes (PLP-DEs) with crucial physiological and pathological roles are known, a global method enabling their cellular profiling is lacking. Here, we demonstrate the utility of a cofactor probe for the identification of human PLP-binding proteins in living cells. Striking selectivity of human pyridoxal kinase led to a customized labeling strategy covering a large fraction of known PLP-binding proteins across various cancer-derived cell lines. Labeling intensities of some PLP-DEs varied depending on the cell type while the overall protein expression levels of these proteins remained constant. In addition, we applied the methodology for in situ screening of PLP-antagonists and unraveled known binders as well as unknown off-targets. Taken together, our proteome-wide method to study PLP-DEs in human cancer-derived cells enables global understanding of the interactome of this important cofactor.

The antimalarial drugs chloroquine and primaquine inhibit pyridoxal kinase, an essential enzyme for vitamin B6 production

Quinoline derivatives such as chloroquine and primaquine are widely used for the treatment of malaria. These drugs are also used for the treatment of trypanosomiasis, and more recently for cancer therapy. However, molecular target(s) of these drugs remain unclear. In this study, we have identified human pyridoxal kinase as a binding protein of primaquine. Primaquine inhibited pyridoxal kinases of malaria, trypanosome and human, while chloroquine inhibited only malaria pyridoxal kinase. Thus, we have identified pyridoxal kinase as a possible target molecule of the antimalarial drugs chloroquine and primaquine.

Catalytic and regulatory roles of species involved in metal–nucleotide equilibriums in human pyridoxal kinase

Pyridoxal 5'-phosphate is the active form of vitamin B6 and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal-nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn(2+) the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP(4-) and HATP(3-) did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP(2-), although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn(2+); as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC50 of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal-nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.

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.

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 ∼2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 Å) shows bound ATP, PL and PLP, while D235N (2.3 Å) 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.

Crystal Structure of human pyridoxal kinase: Structural basis of M+ and M2+ 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.

Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

Pyridoxal kinase (PLK) (EC 2.7.1.35) catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5.-phosphate (PLP), an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B 6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method (GenBank accession number: DQ452397). The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 kDa. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

The human pyridoxal kinase, a plausible target for ginkgotoxin from Ginkgo biloba

Ginkgotoxin (4'-O-methylpyridoxine) occurring in the seeds and leaves of Ginkgo biloba, is an antivitamin structurally related to vitamin B(6). Ingestion of ginkgotoxin triggers epileptic convulsions and other neuronal symptoms. Here we report on studies on the impact of B(6) antivitamins including ginkgotoxin on recombinant homogeneous human pyridoxal kinase (EC 2.7.1.35). It is shown that ginkgotoxin serves as an alternate substrate for this enzyme with a lower K(m) value than pyridoxal, pyridoxamine or pyridoxine. Thus, the presence of ginkgotoxin leads to temporarily reduced pyridoxal phosphate formation in vitro and possibly also in vivo. Our observations are discussed in light of Ginkgo medications used as nootropics.

Crystal structure of human pyridoxal kinase

Pyridoxal kinase, a member of the ribokinase superfamily, catalyzes the ATP-dependent phosphorylation reaction of vitamin B6 and is an essential enzyme in the formation of pyridoxal-5′-phosphate, a key cofactor for over 100 enzymes. Pyridoxal kinase is thus regarded as a potential target for pharmacological agents. In this paper, we report the 2.8 Å crystal structure of human pyridoxal kinase (HPLK) expressed in Escherichia coli. The diffraction data revealed unexpected merohedral perfect twinning along the crystallographic c axis. Taking perfect twinning into account, the structure in dimeric form was well refined according to the CNS program. Structure comparison reveals that the key 12-residue peptide over the active site in HPLK is a β-strand/loop/β-strand flap, while the corresponding peptide in sheep brain enzyme adopts a loop conformation. Moreover, HPLK possesses a more hydrophobic ATP-binding pocket. This structure will facilitate further biochemical studies and structure-based design of drugs related to pyridoxal kinase.

Tat-mediated protein transduction of human brain pyridoxal kinase into PC12 cells

Pyridoxal kinase (PK) catalyses the phosphorylation of vitamin B 6 to pyridoxal-5′-phosphate (PLP). A human brain PK gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat–PK fusion protein. The expressed and purified Tat–PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat–PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B 6, was increased by pre-treatment of Tat–PK to the PC12 cells. Those results suggest that the transduction of Tat–PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B 6.

The Genetic Basis of Human Erythrocyte Pyridoxal Kinase Activity Variation.

Previous studies have shown that there is a striking difference in the erythrocyte activity of the human pyridoxal kinase (PdxK) enzyme, with African-Americans having approximately half the mean activity of their Caucasian counterparts. This difference was observed only in erythrocytes, with leukocyte and skin fibroblast PdxK enzyme activities being similar in both ethnic groups (Science187: 1084–1086, 1975). At the time of the original report it was impossible to elucidate the mechanism by which evolution had selectively lowered the enzyme activity in one cell type but not in another. To now determine the genetic basis for this variation, the entire PdxK gene was sequenced to identify candidate mutations in a sample of Caucasian and African-Americans. A number of variations were discovered, with one interestingly being a deletion located in the promoter region. This deletion was found to occur at a gene frequency of 42.7% in Caucasians (n=56), 25.3% in Asians (n=162) and 56.6% in African-Americans (n=334). Sequence analysis of the PdxK promoter using the software program MatInspector (Nucleic Acids Research23: 4878–4884, 1995) revealed that the deletion removed a putative Core-Promoter Binding Promoter (CPBP) transcription factor binding site. The CPBP transcription factor is known to enhance transcriptional activity by at least four fold and is thought to be a general regulator of TATA box-less genes (Journal of Biological Chemistry272: 9573–9580, 1997). The potential CPBP site was located -300bp to the PdxK start codon and was adjacent to a binding site for the erythroid specific transcription factor, Erythroid Krueppel Like Factor (EKLF). This suggested that the presence of the deletion would cause decreased transcription of PdxK in erythroid precursors only. The presence of the deletion element was found to be significantly associated with a reduced erythrocyte PdxK activity (p=0.0017). Although other modifiers of PdxK activity, such as diet, were found to play a role, the identified promoter deletion allows altered erythrocyte specific transcription to exclusively influence erythrocyte PdxK enzyme activity.

Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue

The gene ( pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin B 6) to pyridoxal phosphate was isolated from Dictyostelium discoideum using insertional mutagenesis. Cells of a pykA gene knockout grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate. Sequencing indicated a gene, with one intron, encoding a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase. After expression of the wild-type gene in Escherichia coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a K m of 8.7 μM for pyridoxal. Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using transformation with the wild-type Dictyostelium gene. Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes.

Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue.

The gene (pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin B(6)) to pyridoxal phosphate was isolated from Dictyostelium discoideum using insertional mutagenesis. Cells of a pykA gene knockout grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate. Sequencing indicated a gene, with one intron, encoding a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase. After expression of the wild-type gene in Escherichia coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a K(m) of 8.7 microM for pyridoxal. Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using transformation with the wild-type Dictyostelium gene. Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes.

Structure of pyridoxal kinase from sheep brain and role of the tryptophanyl residues.

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756-10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.

Human Pyridoxal Kinase: cDNA CLONING, EXPRESSION, AND MODULATION BY LIGANDS OF THE BENZODIAZEPINE RECEPTOR

Peptide fragments of a porcine benzodiazepine-binding protein were used to isolate the cDNA of a related human protein. The cDNA encodes a polypeptide of 312 amino acid residues that is homologous to a bacterial pyridoxal kinase. Transient expression of the cDNA in human embryonic kidney cells confirmed that it encodes human pyridoxal kinase. The recombinant enzyme displayed a Km value of 3.3 microM for pyridoxal and was inhibited competitively by 4-deoxypyridoxine (Ki = 2.8 microM). Benzodiazepine receptor ligands that bound to the purified porcine protein also exerted a potent inhibitory effect on human pyridoxal kinase activity. Transcripts of the pyridoxal kinase gene were detectable in all human tissues examined, and were particularly abundant in the testes. The gene is localized on chromosome 21q22.3 and represents a candidate gene for at least one genetic disorder that has been mapped to this region (autoimmune polyglandular disease type 1).