Brain Pyridoxal Kinase Research Articles

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.

P.703 Administration of thiamine to rats regulates the brain pyridoxal kinase and pyridoxal-5’-phosphate phosphatase without affecting pyridoxine-5’-phosphate oxidase

P.703 Administration of thiamine to rats regulates the brain pyridoxal kinase and pyridoxal-5’-phosphate phosphatase without affecting pyridoxine-5’-phosphate oxidase

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.

Crystal Structure of Brain Pyridoxal Kinase, a Novel Member of the Ribokinase Superfamily

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 A, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B(6) and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp(118) and Tyr(127) in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.

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.

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.

Pyridoxal kinase immunoreactivity in rabbit brain.

Murine polyclonal antibody against purified bovine brain pyridoxal kinase (EC 2.7.1.35) was generated and showed cross-reactivity with rabbit brain pyridoxal kinase. This antibody was used to immunohistochemically examine the distribution of pyridoxal kinase in the rabbit brain. The cytoplasm of neuronal cells and neuroglial cells in the cerebral cortex, hippocampal region, brain nuclei and cerebellar cortex showed positive staining with various degrees of intensity. The neuronal cells and surrounding fibers in some brain nuclei, such as the area tegmentalis ventralis or the substantia nigra, showed intense staining. The neuronal cells of the hippocampal region showed somewhat weak reactivity, but some with intense reactivity were found sparsely distributed and positive staining fiber networks of a very low density were also observed.

Reversible unfolding of pyridoxal kinase.

The unfolding of brain pyridoxal kinase by guanidinium HCl has been investigated at equilibrium. The overall process was reversible as judged from the complete recovery of catalytic activity after removal of guanidinium HCl. Unfolding of pyridoxal kinase was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the spectroscopic changes between 0.2 and 1.5 M guanidinium HCl, and the lack of any discernible plateau suggests that unfolding of the monomer is a cooperative process. A compact intermediate on the unfolding pathway of pyridoxal kinase could not be detected by the method of denaturant gel filtration. The fluorescent analogs of the substrates ATP and pyridoxal were used to assess differences in stability among the domains of the protein. Based on fluorescence and steady emission anisotropy results, it is postulated that the nucleotide domain is more stable than the pyridoxal domain of the kinase.

Purification and characterization of metallothionein and its activation of pyridoxal phosphokinase in trout ( Salmo gairdneri) brain

1. 1. Brain metallothionein was isolated and purified for the first time from rainbow trout. 2. 2. Brain metallothionein exhibited an elution volume ( V e/ V o) of 2.0, had a molecular weight of 6762 Daltons, and contained a zinc content of 9 μg/mg protein. 3. 3. Brain pyridoxal phosphokinase was isolated and assayed for the first time in rainbow trout. 4. 4. Zinc (0.20 μM) or zinc metallothionein (6–30 μM) stimulated the activity of brain pyridoxal kinase in a linear fashion. 5. 5. The results of these studies are interpreted to suggest that in trout brain zinc metallothionein may participate in metabolism of vitamin B 6 and formation of pyridoxal phosphate, the active coenzyme.

Arrangement of the substrates at the active site of brain pyridoxal kinase.

The distances between enzyme-bound paramagnetic CrATP (a stable, beta, gamma-bidentate complex of Cr3+ and ATP) at the active site of sheep brain pyridoxal kinase and the protons of bound inhibitor 4-dPyr (4-deoxypyridoxine) were determined in the ternary enzyme-CrATP.4-dPyr complex by measuring the paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the protons of 4-dPyr. The correlation time for the Cr(3+)-4-dPyr dipolar interaction on the enzyme was estimated as 1.59 ns by the frequency dependence of 1/T1p of water protons. Temperature dependence of 1/T1p values indicated the fast exchange of 4-dPyr from the paramagnetic enzyme.CrATP.4-dPyr complex; hence the measured 1/T1p values can be used for metalnucleus distance determinations. The distances from the Cr3+ of the enzyme-bound CrATP to the 2-methyl (7.19 A), 4-methyl (7.18 A), and H6 proton (6.18 A) of the 4-dPyr are too great to permit a direct coordination of any group from 4-dPyr. However, these distances can be built into a model in which phosphorus of the gamma-phosphoryl group of ATP is 4 A away from the oxygen atom of the 5-CH2OH group of the 4-dPyr. This suggests that phosphorylation of pyridoxal can occur via direct transfer of the phosphoryl group between the bound substrates at the active site of pyridoxal kinase.

Pyridoxal Kinase

Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by HPLC and used for binding studies with ATP and pyridoxal analogues. The spectroscopic properties of trinitrophenyl-ATP bound to the 24-kDa fragment are indistinguishable from those of TNP-ATP bound to the native kinase. The small 16-kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the analogues. The same pattern of digestion was observed when IAF pyridoxal kinase, carrying a fluorescent probe covalently bound to a specific SH residue, was preincubated with chymotrypsin. The kinetics of proteolysis of IAF-pyridoxal kinase was monitored by emission anisotropy, and the analysis of the initial rate of proteolysis at various concentrations of chymotrypsin reveals that the rate of unfolding of native pyridoxal kinase plays a dominant role in the proteolytic process.

Inhibition of Pyridoxal Kinase by Methylxanthines

In the presence of saturating concentrations of adenosine triphosphate (ATP) and rate-limiting amounts of pyridoxal, theophylline was found to inhibit sheep brain pyridoxal kinase (EC 2.7.1.35) competitively. The apparent inhibition constant (Ki) of theophylline for pyridoxal kinase was determined as 8.7 mumol/l. Theophylline concentrations of up to 60 mumol/l did not affect pyridoxal phosphorylation in the presence of saturating amounts of pyridoxal and rate-limiting concentrations of ATP. Caffeine was less potent to inhibit pyridoxal kinase (Ki = 45 mumol/l) due to the presence of a methyl group on the 7 position of the xanthine ring structure. Theobromine showed only a weak inhibition of pyridoxal kinase (Ki = 453 mumol/l). The presence of a hydroxyethyl, hydroxypropyl or dihydroxypropyl group on the N7 position of theophylline completely abolished inhibition of pyridoxal kinase. Enprofylline (3-propylxanthine), a recently described bronchodilator, was also able to inhibit pyridoxal kinase with a Ki of 256 mumol/l.

Activation of pyridoxal kinase by metallothionein

Brain pyridoxal kinase, which uses ATP complexed to either Zn(II) or Co(II) as substrates, displays high catalytic activity in the presence of Zn-thionein and Co-thionein. Several steps intervene in the process of pyridoxal kinase activation, i.e., binding of Zn ions to ATP and interaction between Zn-ATP and the enzyme. Equilibrium binding studies show that ATP mediates the release of Zn ions from the metal-thiolate clusters of the thioneins, whereas spectroscopic measurements conducted on Co-thionein reveal that the absorption transitions corresponding to the metal-thiolate of the protein are perturbed by ATP. The binding Zn-ATP to the kinase proceeds with a ΔG = − 6.3 kcal/mol as demonstrated by fluorometric titrations. Direct interaction between the kinase and derivatized-metallothionein could not be detected by emission anisotropy measurements, indicating that juxtaposition of the proteins does not influence the exchange of metal ions. Since the concentration of free Zn in several mammalian tissues is lower than 1 nM, it is postulated that under in vivo conditions the concentration of metallothionein regulates the catalytic activity of pyridoxal kinase.

Proteolytic cleavage of pyridoxal kinase into two structural domains

Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields 2 fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by chromatographic techniques and analyzed for interaction with the ATP analogue, trinitrophenyl-ATP, using fluorescence spectroscopy. The absorption and fluorescence properties of trinitrophenyl-ATP bound to the fragment of 24 kDa (emission maximum, 540 nm, emission anisotropy at 460 nm, 0.30, and fluorescence lifetime, γ = lns) are indistinguishable from those of the ATP analogue bound to the native enzyme. The fragment of 16 kDa does not bind trinitrophenyl-ATP. The results are consistent with the hypothesis that monomeric pyridoxal kinase is folded into 2 domains connected by a single polypeptide chain sensitive to proteolytic cleavage.

Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site

4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.

Brain pyridoxal kinase dissociation of the dimeric structure and catalytic activity of the monomeric species.

Reversible dissociation of the dimeric structure of brain pyridoxal kinase into subunits was attained by addition of guanidinium HCl (2 M). The molecular mass of the subunits (40 kDa) was determined by HPLC chromatography. Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi-gel 15). The matrix-bound monomer is catalytically competent. The reaction of the crosslinking reagent 4,4'-dimaleimidestilbene 2,2'-disulfonate (DMDS), a derivatized stilbene, with the dimeric structure of pyridoxal kinase resulted in the formation of an oligomeric species of 80 kDa detectable by SDS-PAGE. The crosslinked subunits exhibit the same catalytic parameters as the native enzyme. The presence of two nucleotide-binding sites per dimer was determined by fluorimetric titrations using pyridoxyl-ATP, a strong competitive inhibitor with respect to ATP. The ATP analog binds with a Kd = 5 microM to each nucleotide site of the dimeric enzyme. The mode of binding pyridoxyl-ATP to the kinase is discussed in reference to a model which assumes the presence of two binding domains per subunit.

Sheep brain pyridoxal kinase: fluorescence spectroscopy of the dimeric enzyme

Pyridoxal kinase (ATP:pyridoxal 5-phosphotransferase, EC 2.7.1.35) has been purified 9000-fold from sheep brain by affinity chromatography. The enzyme of 80 000 molecular weight is made up of two identical-size subunits. The interaction of the inhibitor N-dansyl-1,8-diaminooctane with the nucleotide site of the kinase was examined by means of steady and nanosecond fluorescence spectroscopy. N-Dansyl-1,8-diaminooctane is a competitive inhibitor with respect to AtP at saturating concentrations of pyridoxal. It binds to the nucleotide site of the enzyme with K d = 2.2 μM. Bound N-dansyl-1,8-diaminooctane is shielded from collisional encounters with the external quencher acrylamide. The collisional rate constant for bound N-dansyl-1,8-diaminooctane ( K q = 1.4 · 10 8 M −1 · s −1) is 10-times lower than the value obtained for the free chromophore. Nanosecond emission anisotropy measurements yield a rotational correlation time of 42 ns for the inhibitor complexes to the kinase. Both steady and nanosecond fluorescence results are consistent with a model in which the inhibitor bound to the nucleotide site is immobilized by amino acids located at the catalytic site.

Brain pyridoxal kinase. Purification and characterization.

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.

Nucleoside phosphorothioates as probes of the nucleotide binding site of brain pyridoxal kinase.

N-Dansyl-2-oxopyrrolidine, a competitive inhibitor with respect to ATP, was used as a probe of the nucleotide binding site of pyridoxal kinase. It binds to an hydrophobic region of the catalytic site with a KD = 6 microM. Time emission anisotropy measurements yielded a rotational correlation time of 38 ns for the bound inhibitor. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the nucleotide binding site. Protein fluorescence quenching was used to determine the dissociation constants of the diastereomers of ATP beta S. Both diastereomers bind with a dissociation constant KD = 25 microM. The kinetic parameters Km and Vmax for the pyridoxal kinase reaction were determined for ATP and the diastereomers of ATP beta S in the presence of Mg(II), Co(II), Zn(II), and Cd(II). With Mg(II), pyridoxal kinase exhibits stereoselectivity for the A diastereomer of ATP beta S, Vmax ratio, A/B = 30. In the presence of Cd(II), the stereoselectivity is reversed and the B diastereomer of ATP beta S is the preferred substrate. As the divalent cations were varied in the series Mg(II), Co(II), Zn(II), and Cd(II), the A/B ratio was progressively lowered to the value of 0.2 found for Cd(II). These data indicate that the divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrates. The results obtained with the diastereomers of ATP alpha S suggest that pyridoxal kinase uses the divalent cation, delta,beta,gamma-bidentate nucleotide chelate as substrate.