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.     

Affinity labeling of pyridoxal kinase with adenosine polyphosphopyridoxal

Pyridoxal kinase is inactivated by preincubation with the affinity label reagent adenosine tetraphosphate pyridoxal (AP4-PL) at a mixing molar ratio of 5:1 AP4-PL contains structural features of the substrates pyridoxal and ATP. The substrate ATP affords substantial protection against inactivation. The extent of chemical modification by the affinity label was determined by measuring the spectroscopic properties of AP4-pyridoxyl chromophores attached to the enzyme after reduction with NaBH4. The incorporation of 2 mol of the affinity label per enzyme dimer is needed for complete inactivation of the kinase. After chymotryptic digestion of the enzyme modified with AP4-PL and reduced with tritiated NaBH4, only one radioactive peptide absorbing at 325 nm was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysyl residue of monomeric pyridoxal kinase has reacted with the affinity label reagent. It is postulated that the modified lysyl residue is involved in direct interactions with phosphoryl groups of ATP.     

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.     

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.     

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.     

Human pyridoxal kinase: overexpression and properties of the recombinant enzyme

Pyridoxal kinase catalyses the phosphorylation of the vitamin B6. A human brain pyridoxal kinase cDNA was isolated, and the recombinant enzyme was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). Pure pyridoxal kinase exhibits a molecular mass of about 40 kDa when examined by SDS-PAGE and FPLC gel filtration. The recombinant enzyme is a monomer endowed with catalytic activity, indicating that the native quaternary structure of pyridoxal kinase is not a prerequisite for catalytic function. Zn2+ is the most effective divalent cation in the phosphorylation of pyridoxal, and the human enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates pyridoxal and ATP are 97 microM and 12 microM, respectively. In addition, the unfolding processes of the recombinant enzyme were monitored by circular dichroism. The values of the free energy change of unfolding (AGo = 1.2 kcal x mol(-1) x K(-1)) and the midpoint transition (1 M) suggested that the enzyme is more stable than ovine pyridoxal kinase against denaturation by guanidine hydrochloride. Intrinsic fluorescence spectra of the human enzyme from red-edge excitation and fluorescence quenching experiments showed that the tryptophanyl residues are not completely exposed and more accessible to neutral acrylamide than to the negatively charged iodide. The first complete set of catalytic and structural properties of human pyridoxal kinase provide valuable information for further biochemical studies on this enzyme.     

Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine.

Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.     

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.     

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.     

Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog.

The crystal structure of the phosphorylated, activated form of the insulin receptor tyrosine kinase in complex with a peptide substrate and an ATP analog has been determined at 1.9 A resolution. The activation loop (A-loop) of the kinase undergoes a major conformational change upon autophosphorylation of Tyr1158, Tyr1162 and Tyr1163 within the loop, resulting in unrestricted access of ATP and protein substrates to the kinase active site. Phosphorylated Tyr1163 (pTyr1163) is the key phosphotyrosine in stabilizing the conformation of the tris-phosphorylated A-loop, whereas pTyr1158 is completely solvent-exposed, suggesting an availability for interaction with downstream signaling proteins. The YMXM-containing peptide substrate binds as a short anti-parallel beta-strand to the C-terminal end of the A-loop, with the methionine side chains occupying two hydrophobic pockets on the C-terminal lobe of the kinase. The structure thus reveals the molecular basis for insulin receptor activation via autophosphorylation, and provides insights into tyrosine kinase substrate specificity and the mechanism of phosphotransfer.     

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.     

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.     

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.     

Activation and inactivation of horse liver alcohol dehydrogenase with pyridoxal compounds.

Pyridoxal compounds can either activate or inactivate horse liver alcohol dehydrogenase in differential labeling experiments. Amino groups outside of the active sites were modified with ethyl acetimidate, while the amino groups in the active sites were protected by the formation of the complex with NAD-plus and pyrazole. After removal of the NAD-plus and pyranzole, the partially acetimidylated enzyme was reductively alkylated with pyridoxal and NaBH4, with the incorporation of one pyridoxal group per subunit of the enzyme. The turnover numbers for the reaction of NAD-plus and ethanol increased by 15-fold, and for NADH and acetaldehyde by 32-fold. The Michaelis and inhibition constants increased 80-fold or more. Pyridoxal phosphate and NaBH4 also modified one group per subunit, but the turnover numbers decreased by 10-fold and the kinetic constants were intermediate between those obtained for pyridoxyl alcohol dehydrogenase and the partially acetimidylated enzyme. With native enzyme, the rates of dissociation of the enzyme-coenzyme complexes are rate-limiting in the catalytic reactions. The pyridoxyl enzyme is activated because the rates of dissociation of the enzyme-coenzyme complexes are increased. The rates of binding of coenzyme to phosphopyridoxyl enzyme have decreased due to the introduction of the negatively charged phosphate. The size of the group is not responsible for this decrease since these rates are not greatly decreased by the incorporation of pyridoxal. For both pyrodoxal and phosphopyridoxyl alcohol dehydrogenases, the interconversion of the ternary complex is at least partially rate-limiting. Chymotryptic-tryptic digestion of pryidoxyl enzyme produced a major peptide corresponding to residues 219 to 229, in which Lys 228 had reacted with pyridoxal. The same lysine residue reacted with pyridoxal phosphate.     

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.     

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.     

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

Pyridoxal‐5′‐phosphate (vitamin B₆) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B₆de novo and rely on uptake of B₆ 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.     

Inhibition of the recBC enzyme of Escherichia coli by specific binding of pyridoxal 5'-phosphate to DNA binding site.

Pyridoxal 5'-phosphate rapidly abolished the DNA-hydrolyzing activities as well as DNA-dependent ATP-ase activity of the recBC enzyme of Escherichia coli. Pyridoxal also had an inhibitory effect on the enzyme but less effective than that of pyridoxal 5'-phosphate. Pyridoxamine 5'-phosphate, pyridoxamine, or pyridoxine had no effect on the activities of the enzyme. The inhibition was rapidly reversed by dilution but could be made irreversible by reduction with sodium borohydride prior to dilution. This suggests the formation of Schiff base between pyridoxal 5'-phosphate and an epsilon-amino group of a lysine residue which is essential for the enzyme activity. Pyridoxal 5'-phosphate is a competitive inhibitor of DNA substrate but not of ATP. Furthermore, the presence of DNA substrate protected the enzyme from inactivation by the reduction but the presence of ATP showed no effect. Thus, the recBC enzyme appears to have an essential lysine residue at or near the DNA binding site of the enzyme, and the enzyme possesses two independent catalytic sites, such as a DNA binding site and an ATP binding site.     

The crystal structure of SDR-type pyridoxal 4-dehydrogenase of Mesorhizobium loti

Pyridoxal 4-dehydrogenase catalyzes the irreversible oxidation of pyridoxal to 4-pyridoxolactone and is involved in degradation pathway I of pyridoxine, a vitamin B(6) compound. Its crystal structure was elucidated for the first time. Molecular replacement with (S)-1-phenylthanol dehydrogenase (PDB code 2EW8) was adopted to determine the tertiary structure of the NAD(+)-bound enzyme.