Correlative changes of pyridoxal kinase pyridoxal-5'-phosphate and glutamate decarboxylase in brain, during drug-induced convulsions.
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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 angstroms 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 beta-strand/loop/beta-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.
Adenosine Triphosphate↗
Expression, purification, and kinetic constants for human and Escherichia coli pyridoxal kinases.
Pyridoxal kinase is an ATP dependent enzyme that phosphorylates pyridoxal, pyridoxine, and pyridoxamine forming their respective 5'-phosphorylated esters. The kinase is a part of the salvage pathway for re-utilizing pyridoxal 5'-phosphate, which serves as a coenzyme for dozens of enzymes involved in amino acid and sugar metabolism. Clones of two pyridoxal kinases from Escherichia coli and one from human were inserted into a pET 22b plasmid and expressed in E. coli. All three enzymes were purified to near homogeneity and kinetic constants were determined for the three vitamin substrates. Previous studies had suggested that ZnATP was the preferred trinucleotide substrate, but our studies show that under physiological conditions MgATP is the preferred substrate. One of the two E. coli kinases has very low activity for pyridoxal, pyridoxine, and pyridoxamine. We conclude that in vivo this kinase may have an alternate substrate involved in another metabolic pathway and that pyridoxal has only a poor secondary activity for this kinase.
Adenosine Triphosphate↗
Cloning and characterization of Arabidopsis thaliana pyridoxal kinase.
Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis. Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.
Amino Acid Sequence↗
Roscovitine targets, protein kinases and pyridoxal kinase.
(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.
Adenosine Triphosphate↗
Interaction between pyridoxal kinase and pyridoxal-5-phosphate-dependent enzymes.
The interactions of two pyridoxal-5-phosphate (PLP)-dependent enzymes, alanine aminotransferase (ALT) and glutamate decarboxylase (GAD), with pyridoxal kinase (PK) were studied by fluorescence polarization as well as surface plasmon resonance techniques. The results demonstrated that PK can specifically bind to ALT and GAD. Moreover, binding profiles of both enzymes to immobilized PK were altered by excess amount of PLP. The equilibrium affinity constants for ALT in the absence and presence of PLP are 20.4 x 10(4) M(-1)and 6.7 x 10(4) M(-1), and for GAD are 37 x 10(4) M(-1)and 20.8 x 10(4) M(-1), respectively. It appears that specific interactions occur between PK and PLP-dependent enzymes, and the binding affinities of PK for PLP-dependent enzymes decrease in the presence of PLP. The results support our hypothesis that PLP transfer from PK to PLP-dependent enzymes requires a specific interaction between PK and the enzyme.
Alanine Transaminase↗
Inactivation of microbial pyridoxal kinase by pyridoxal.
Pyridoxal kinase from Escherichia coli and bakers' yeast was inactivated by pyridoxal while the enzyme from rat and pig brain was not. The inactivation of the enzyme purified from E. coli was reversible and was rendered irreversible by the reduction with NaBH4. This finding as well as a similar inactivation by 5'-deoxypyridoxal but not by 4'-deoxypyridoxine suggested that the inactivation was due to Schiff base formation. The suggestion was confirmed by the incorporation of tritium label into the enzyme by the reaction of the enzyme with [3H] pyridoxal followed by the treatment with NaBH4. Correlation between the loss of enzyme activity and the amount of pyridoxal bound to the enzyme showed that binding of pyridoxal to one crucial site completely inactivated the enzyme. Pyridoxine and 4'-deoxypyridoxine did not have a protective effect against inactivation indicates that the binding site was not the substrate site. The results of kinetic and equilibrium analyses were consistent with a one-step inactivation mechanism.
Animals↗
Crystal structure of pyridoxal kinase from the Escherichia coli pdxK gene: implications for the classification of pyridoxal kinases.
The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-A resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4' substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4' of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.
Adenosine Triphosphate↗
[Pyridoxal kinase activity and pyridoxal-P concentration in mammalian tissues under normal and experimental conditions].
The activity and the distribution of pyridoxal kinase in rat and mouse tissues are studied. The data obtained testify the presence of a relative excess of pyridoxal kinase in all the organs studied, which probably causes a high rate of pyridoxalphosphate (PLP) biosynthesis under comparatively low vitamin B6 concentration. A correlation between the level of pyridoxal kinase activity and the content of PLP in rat brain and liver during ontogenesis is observed. The activity of pyridoxal kinase and the content of PLP are shown to be sharply increased in liver of rats received a protein-rich diet. Bilateral adrenalectomy resulted in the decrease of absolute and specific enzyme activities in rat liver by 20--30%. The content of PLP in mouse brain and liver was sharply decreased under experimental B6-avitaminosis while the content of pyridoxal kinase practically did not change. The injection of vitamin B6 rapidly normalized the PLP content in mouse tissues. The data obtained show that under physiological conditions the functional activity of pyridoxal kinase may be regulated in tissues by enzyme and substate contents. Some aspects of vitamin B6 metabolism in mammals are considered. It is concluded that in body the pyridoxal catabolism connected with its phosphorylation by pyridoxal kinase and the formation of pyridoxalphosphate.
Adrenalectomy↗
Modulation of the catalytic activity of pyridoxal kinase by metallothionein.
Pyridoxal kinase displays high catalytic activity in the presence of metallothionein. The apoprotein of metallothionein as well as the peptide LYS-CYS-THR-CYS-CYS-ALA exert a strong inhibitory effect upon pyridoxal kinase by sequestering free Zn ions. Several steps intervene in the process of pyridoxal kinase activation, i.e. binding of Zn ions by ATP and interaction of Zn-ATP with the enzyme; but direct interaction between metallothionein and pyridoxal kinase (protein association) could not be detected by emission anisotropy measurements. Since the concentration of free Zn++ in mammalian tissues is lower than 10(-9)M, it is postulated that the concentration of metallothionein regulates the catalytic activity of pyridoxal kinase. The mechanism of reconstitution of the metalloenzyme yeast aldolase in the presence of metallothionein was also investigated.
Adenosine Triphosphate↗
Brain pyridoxal kinase. Mobility of the substrate pyridoxal and binding of inhibitors to the nucleotide site.
Pyridoxal kinase has been purified 2000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N-dansyl-2-oxopyrrolidine (dansyl = 5-dimethylaminonaphthalene-1-sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd = 11 microns at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N-Dansyl-2-oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 microns. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N-dansyl-2-oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.
Animals↗
Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis.
Pyridoxal kinases (PdxK) are able to catalyse the phosphorylation of three vitamin B(6) precursors, pyridoxal, pyridoxine and pyridoxamine, to their 5'-phosphates and play an important role in the vitamin B(6) salvage pathway. Recently, the thiD gene of Bacillus subtilis was found to encode an enzyme which has the activity expected of a pyridoxal kinase despite its previous assignment as an HMPP kinase owing to higher sequence similarity. As such, this enzyme would appear to represent a new class of ;HMPP kinase-like' pyridoxal kinases. B. subtilis thiD has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a binary complex with ADP and Mg(2+). X-ray diffraction data have been collected from crystals to 2.8 A resolution at 100 K. The crystals belong to a primitive tetragonal system, point group 422, and analysis of the systematic absences suggest that they belong to one of the enantiomorphic pair of space groups P4(1)2(1)2 or P4(3)2(1)2. Consideration of the space-group symmetry and unit-cell parameters (a = b = 102.9, c = 252.6 A, alpha = beta = gamma = 90 degrees ) suggest that the crystals contain between three and six molecules in the asymmetric unit. A full structure determination is under way to provide insights into aspects of the enzyme mechanism and substrate specificity.
Adenosine Diphosphate↗
Crystallization and preliminary crystallographic studies of pyridoxal kinase from sheep brain.
Pyridoxal kinase (ATP:pyridoxal 5'-phosphotransferase; EC 2.7.1.35) is a key enzyme in the transformation of vitamin B(6) to pyridoxal-5'-phosphate. Pyridoxal-5'-phosphate is the crucial cofactor required by numerous enzymes involved in the metabolism of amino acids and the synthesis of many neurotransmitters. Pyridoxal kinase from sheep brain was crystallized in an orthorhombic form using the hanging-drop vapour-diffusion method with sodium citrate as the precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 59.8, b = 94.4, c = 128.2 A, and diffract to a resolution of 2.1 A. Crystals were transferred into a soaking liquid without citrate and two heavy-atom derivatives were prepared.
Animals↗
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 delta G = -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.
Adenosine Triphosphate↗
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.
Animals↗
Purification and characterization of pyridoxal kinase from human erythrocytes.
Pyridoxal kinase has been purified 50,000-fold from human erythrocytes. The purification procedure included dextran-induced aggregation of red blood cells, ammonium sulphate fractionation of the haemolysate, DEAE-cellulose chromatography, hydroxyapatite chromatography. Sephadex G-100 gel filtration and omega-aminooctyl agarose chromatography. The enzyme preparation migrated as a single protein and activity band on analytical gel electrophoresis. Determination of the Michaelis constants for pyridoxal, pyridoxine and pyridoxamine using a new assay gave comparable values of 33 microM, 16 microM and 6.2 microM respectively. Various amines were shown as competitive inhibitors of pyridoxal kinase with respect to ATP. The inhibition order was: N-dansyl-1,8-diaminooctane greater than 1,8-diaminooctane greater than 1,6-diaminohexane greater than 1,4-diaminobutane greater than gamma-aminobutyric acid, whereas octane, hexane and butane were not inhibitors. Results suggest that the amino groups on the above inhibitors are essential for competitive inhibition at saturating concentrations of pyridoxal. It was also observed that increasing the chain length of the hydrophobic backbone of these competitive inhibitors can facilitate its action.
Catalysis↗
Characterization of Trypanosoma brucei pyridoxal kinase: purification, gene isolation and expression in Escherichia coli.
Pyridoxal kinase catalyzes the ATP-dependent phosphorylation of vitamin B6, generating pyridoxal-5'-phosphate, an important cofactor for many enzymatic reactions. Pyridoxal kinase was purified 4300-fold to homogeneity from Trypanosoma brucei and peptides generated by proteolysis were subjected to amino acid sequence analysis. The peptide sequence information was used to generate a partial clone of T. brucei pyridoxal kinase by polymerase chain reaction (PCR), which in turn was used to screen a T. brucei genomic library for a full length clone. The 903-bp gene was sequenced and found to encode a 300-amino acid protein. The deduced amino acid sequence contains all of the peptide sequences obtained from the proteolytic cleavage of the native enzyme and shares 28% sequence identity with a putative Escherichia coli pyridoxal kinase, identified for its ability to compliment pyridoxal kinase deficient cells. The T. brucei pyridoxal kinase gene was expressed in E. coli and the purified enzyme was found to have pyridoxal kinase activity, confirming that this gene encodes the functional T. brucei enzyme. Native and recombinant pyridoxal kinase have a monomer molecular weight of 37 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are dimers in solution. Native T. brucei pyridoxal kinase catalyzes the phosphorylation of pyridoxal with a specific activity of 990 nmol min(-1) per mg and apparent Km values for pyridoxal and ATP of 22 and 9 microM. respectively. Substrate inhibition is observed for pyridoxal. Similar results were obtained for the recombinant enzyme.
Amino Acid Sequence↗
Inhibition of pyridoxal kinase by the pyridoxal-gamma-aminobutyrate imine.
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Amino Acids↗