Isolation and characterization of the 5'-flanking region of the human PDXK gene.

Pyridoxal kinase is a key enzyme for the biosynthesis of pyridoxal 5'-phosphate. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B6, and acts as a cofactor in >140 different enzyme reactions. It is still unknown how the kinase synthesis is regulated in the cells, and nothing has been reported about the gene promoter. In the present study, based on the bioinformatics analysis of the 5'-flanking region of the human PDXK gene, we cloned the promoter region by PCR. Through the construction of a series of luciferase expression vectors containing the human PDXK promoter region, we characterized the promoter in terms of its structure and function. The transcription start site is at 198bp upstream of the ATG translation initiation site. An important regulatory region is located at -665/-433bp upstream of the transcription start site. The promoter lacks the canonical TATA box, but contains three GC-boxes and one E-box. A deletion and mutation experiment revealed that the transcription factor Sp1 binding site C (-553/-543) is critical in maintaining the robust promoter activity. Knockdown of Sp1 by RNA interference and chromatin immunoprecipitation analysis further proved that the Sp1 is involved in the regulation of the PDXK gene expression.

Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

Vitamin B6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B6 vitamers were detected as compared with the control.

Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks.

Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.

Enzymatic conversion from pyridoxal to pyridoxine caused by microorganisms within tobacco phyllosphere.

Vitamin B6 (VB6) comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate (PLP) is a coenzyme involved in a high diversity of biochemical reactions. In plants, PLP is de novo synthesized, and pyridoxine (PN) is usually maintained as the predominant B6 vitamer. Although the conversion from pyridoxal (PL) to PN catalyzed by PL reductase in plants has been confirmed, the enzyme itself remains largely unknown. We previously found pre-incubation at 35 °C dramatically enhanced PL reductase activity in tobacco leaf homogenate. In this study, we demonstrated that the increase in the reductase activity was a consequence of phyllosphere microbial proliferation. VB6 was detected from tobacco phyllosphere, and PL level was the highest among three non-phosphorylated B6 vitamers. When the sterile tobacco rich in PL were kept in an open, warm and humid environment to promote microorganism proliferation, a significant change from PL to PN was observed. Our results suggest that there may be a plant-microbe interaction in the conversion from PL to PN within tobacco phyllosphere.

Enzymatic transamination of pyridoxamine in tobacco plants.

Vitamin B6 (VB6) comprises a group of pyridine compounds that are involved in a surprisingly high diversity of biochemical reactions. Humans and animals depend largely on plants for their VB6 nutrition. Many studies have focused on biosynthesis of VB6 and comparatively little is known about VB6 metabolic conversion in plants. Recently, we have found that an efficient conversion pathway between pyridoxal (PL) and pyridoxamine (PM) is present in tobacco, but the catalytic enzyme remains an unsolved mystery. In this study, enzymes catalyzing the transamination of PM were purified from tobacco leaves and characterized. Our results suggest that a specific PM-pyruvate aminotranferase dominates the reversible transamination of PM in tobacco, and also show that the apo form of glutamic-oxaloacetic aminotranferase from tobacco, but not the holoenzyme, is able to catalyze the analogous transamination reaction between PM and either oxaloacetate or α-ketoglutarate. PM-pyruvate aminotranferase is involved in a degradation pathway for VB6 compounds in bacteria. Therefore, our study raises questions about whether the degradation pathway of VB6 exists in plants.

Effect of abiotic stress on the abundance of different vitamin B6 vitamers in tobacco plants.

There are six different vitamin B6 (VB6) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP), and pyridoxine 5'-phosphate (PNP), of which PLP is the active form. Although transcriptional regulation of the genes involved in the de novo and salvage pathways of PLP syntheses after stress treatments has been described for Arabidopsis thaliana and tobacco plants, it remains open as to whether this in turn affects VB6 levels. In this study, the effects of chilling, UV radiation, intensity of illumination, osmotic pressure, oxidative and drought stresses on the abundance of different B6 vitamers in tobacco plants were examined by using high performance liquid chromatography (HPLC). The abiotic stressors resulted in significant increase of PLP, and caused some corresponding changes with PL and PN. The highest increase of PLP was 2.5-fold compared to the control plants, followed by a continuous decline back to the control levels. These changes are presumably caused by the regulation and control mechanism on the VB6 metabolism in plants.

Characterization of an acid phosphatase responsible for hydrolysis of pyridoxal 5'-phosphate in tobacco plants.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B(6), is an important cofactor for many enzymatic reactions. PLP is also a very reactive molecule, and the hydrolysis of PLP is crucial for controlling intracellular PLP concentrations. However, little is known about the enzymatic hydrolysis of PLP in plants. In this study, a novel acid phosphatase was purified from tobacco leaves and characterized by using PLP as a substrate. This phosphatase was purified 180-fold with a yield of 28% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose FF, Sephadex G-100 and SP Sephadex C-25. Our data revealed that the purified enzyme was a dimer with a molecular mass of approximately 50 kDa. The purified phosphatase had maximum catalytic activity at pH 5.5, and displayed optimal activity at 50 °C. The enzyme required divalent metal ion for activity, and Mg(2+), among a few tested cations, was the most effective for catalysis under saturating substrate concentrations. The activity of the purified phosphatase was inhibited by molybdate, fluoride and EDTA, but was not inhibited by levamisole and tartrate. The phosphatase hydrolyzed a broad range of substrates at different rates, and the hydrolysis of PLP was competitively inhibited by ATP, pNPP, and by the reaction products, PL and inorganic phosphate. The phosphatase had a Km of 0.24 mM and a Vmax of 2.76 µmol/min/mg with PLP. When pyridoxamine 5'-phosphate or pyridoxine 5'-phosphate was tested as a substrate, the phosphatase activity was reduced by 50%. Our study suggests that the enzyme is a nonspecific acid phosphatase responsible for hydrolysis of all three phosphorylated B(6) vitamers in tobacco plants.

Functional significance of some particular amino acid residues in Bombyx mori pyridoxal kinase.

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B(6) metabolism in animals. It catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal 5'-phosphate, an important cofactor for many enzymatic reactions. Bombyx mori PLK (BmPLK) is 10 or more residues shorter than mammalian PLKs, and some amino acid residues conserved in the PLKs from mammals are not maintained in the protein. Multiple sequence alignment suggested that amino acid residues Thr(47), Ile(54), Arg(88), Asn(121) and Glu(230) might play important roles in BmPLK. In this study, we used a site-directed specific mutagenesis approach to determine the functional significance of these particular amino acid residues in BmPLK. Our results demonstrated that the mutation of Asn(121) to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr(47) to Asn, Ile(54) to Phe, and Arg(88) to Ile displayed a decreased catalytic efficiency and an elevated Km value for substrate relative to the wild-type value, and no enzyme activity could be detected in mutant of Trp(230) to Glu. Circular dichroism analysis revealed that the mutation of Trp(230) to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp(230) is crucial to maintain the structural and functional integrity of BmPLK. This study will add to the existing understanding of the characteristic of structure and function of BmPLK.

Characterization of enzymes involved in the interconversions of different forms of vitamin B(6) in tobacco leaves.

There are six different vitamin B(6) (VB(6)) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP) and pyridoxine 5'-phosphate (PNP). PLP is a coenzyme required by more than 100 cellular enzymes. In spite of the importance of this vitamin, the understanding of VB(6) metabolic conversion in plants is limited. In this study, we developed a sensitive and reliable method to assay VB(6)-metabolizing enzyme activities by monitoring their products visually using high-performance liquid chromatography. With this method, the reactions catalyzed by PL/PM/PN kinase, PMP/PNP oxidase, PM-pyruvate aminotransferase, PL reductase and PLP phosphatase were all nicely detected using crude protein extracts of tobacco leaves. Under optimal in vitro conditions, specific activities of those enzymes were 0.15 ± 0.03, 0.10 ± 0.03, 0.08 ± 0.02, 0.64 ± 0.13 and 23.08 ± 1.98 nmol product/min/mg protein, respectively. This is the first report on the conversion between PM and PL catalyzed by PM-pyruvate aminotransferase in plants. Furthermore, the PL reductase activity was found to be heat inducible. Our study sheds light on the VB(6) metabolism taking place in plants.

Interconversions of different forms of vitamin B6 in tobacco plants.

There are six different vitamin B(6) (VB(6)) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP), and pyridoxine 5'-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB(6) in the human diet, and VB(6) plays an important role in plants, the mechanisms underlying the interconversions of different VB(6) forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100mg/L of PM, PL or PN and the abundance of the different B(6) vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB(6) was about 3.9 µg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB(6) by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B(6) vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.

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 B6 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.