JP4675134B2 - Sugar nucleotide synthase and use thereof - Google Patents

Description

  The present invention relates to a novel enzyme that catalyzes the synthesis of important sugar nucleotides in plants, DNA encoding the same, and use thereof.

  There are various oligosaccharides and proteins bound with oligosaccharides in cells, and they are involved in many physiological functions. These molecules are involved in plant cell differentiation and growth control, and the role played by oligosaccharides, polysaccharides, and glycoproteins including pentoses such as arabinose (Ara) and xylose (Xyl) has attracted attention. Sugar nucleotides such as UDP-sugar are involved in the regulation of synthesis and degradation of oligosaccharides having these physiological activities.

  In the synthesis of UDP-sugar, pyrophosphorylase is involved, and in the synthesis of UDP-glucose (UDP-Glc), UDP-Glc pyrophosphorylase is used, and UDP-N-acetylglucosamine (UDP-GlcNAc) is used. UDP-GlcNAc pyrophosphorylase is used for the synthesis of). For example, UDP-Glc pyrophosphorylase was extracted from potato and cDNA was also cloned (see Non-Patent Documents 1 and 2). In addition, UDP-GlcNAc pyrophosphorylase has been identified from pigs and Giardia protozoa (see Non-Patent Documents 3 and 4). It has also been reported that UDP-GlcNAc pyrophosphorylase has a common catalytic site sequence motif (see Non-Patent Document 5), and UDP-GlcNAc pyrophosphorylase from E. coli has two glucosamine monophosphate acetyltransferase activities. It has also been clarified that it is a functional enzyme (see Non-Patent Document 6). Moreover, it has been shown from crystal structure analysis that the catalytic site sequence motif binds to UDP-GlcNAc in the E. coli enzyme (see Non-Patent Document 7). These UDP-GlcNAc pyrophosphorylases are active against hexose monophosphates such as UDP-GlcNAc and UDP-Glc, but have no activity against pentose monophosphate. L-arabinose (L-Ara), a pentose, is a monosaccharide widely distributed in the plant kingdom, and is widely present as a sugar chain component of polysaccharides and proteoglycans constituting plant cell walls. Cell wall components containing L-Ara as the main constituent monosaccharide include arabinan side chain of pectin, arabinogalactan, glucuronoarabinoxylan, extensin, and arabinogalactan-protein (see Non-Patent Documents 8 and 9). These polysaccharides containing L-Ara are considered to be physiologically active substances that play an important role in growth and differentiation.

Sowokinos et al., Plant Physiol., 101, 1073-1080 (1993) Katsube et al., J. Biochem., 108, 321-326 (1990) Szumilo et al., J. Biol.chem., 271, 13147-13154 (1996) Bulik et al., Biochem. Parasitol., 95, 135-139 (1998) Mio et al., J. Biol.chem., 273, 14392-14397 (1998) Mengin-Lecreulx et al., J. Bacteriol., 176, 5788-5795 (1994) Brown et al., The EMBO J., 18, 4096-4107 (1999) Carpita and Gibeaut, Plant J., 3, 1-30 (1993) Showalter, Plant Cell, 5, 9-23 (1993)

  Thus, polysaccharides containing pentose have various physiological activities and are useful, and it is necessary to elucidate their metabolism, biosynthetic mechanism, and regulatory action, but arabinose monophosphate (L-Ara 1-P ) And sugar nucleotide pyrophosphorylase acting on xylose monophosphate (Xyl 1-P), there is almost no research report, and elucidation of the enzyme is desired. UDP-Xylospirophosphorylase (UDP-Xyl pyrophosphorylase, EC2.7.7.11) was extracted from mang bean (Ginsburg V. etal, Pro. Natl. Acad. Sci. USA 42, 333-335, 1956), no knowledge of the isolated enzyme, no knowledge of the amino acid sequence, and no cDNA cloning. There is no EC number for UDP-L-arabinose pyrophosphorylase (UDP-L-Ara pyrophosphorylase). Therefore, it cannot be said that an enzyme that efficiently synthesizes these pentose-nucleotides has been found so far.

As a result of repeated studies to analyze an enzyme that efficiently synthesizes pentose-nucleotide, the present inventors have developed a novel enzyme that efficiently synthesizes UDP-L-Ara from L-Ara 1-P. It was isolated from the extract and analyzed for its characteristics, and the amino acid sequence of the enzyme and the DNA encoding the enzyme could be isolated to reveal the base sequence. Furthermore, the polypeptide chain encoded by the obtained cDNA can be confirmed to have UDP-L-Ara pyrophosphorylase activity and industrial production of enzyme protein having UDP-L-Ara pyrophosphorylase activity is possible. The present invention has been reached as a pioneering property.
That is, the present invention provides a polypeptide and / or enzyme protein having pyrophosphorylase activity and a DNA encoding the same.

  According to the present invention, the characteristics, amino acid sequence, and cDNA sequence of UDP-L-arabinose pyrophosphorylase that has not been known so far can be clarified, and it has been clarified that the protein is a novel protein. According to the present invention, it has become possible to efficiently produce UDP-L-Ara.

Hereinafter, the present invention will be described in detail.
In general, there are two synthetic pathways for producing sugar nucleotides in plants: a de novo pathway synthesized by a conversion reaction of a precursor sugar nucleotide and a salvage pathway synthesized from a free monosaccharide via a phosphorylation reaction. In the Salvage pathway, UDP-L-Ara is thought to be synthesized from free L-Ara via Ara 1-P to UDP-L-Ara by UDP-L-Ara pyrophosphorylase (Feingold, The Biochemistry of Plants Vol. 3, 101-170, Academic Press, New York, (1980)). Since oligosaccharides containing L-Ara are widely present in plants, it is speculated that an enzyme involved in their synthesis, namely UDP-L-Ara pyrophosphorylase, is present in plant cells. However, the enzyme has not been isolated so far, and the properties of the protein molecule, amino acid sequence, and encoding DNA sequence were not known at all.

  The enzyme protein having UDP-L-Ara pyrophosphorylase activity of the present invention (hereinafter also referred to as this enzyme) is a molecule that acts on Ara 1-P and catalyzes the synthesis of UDP-L-Ara. In the present invention, including protein molecules obtained by extraction from plants, polypeptide chains excised from protein molecules, proteins and polypeptide chains expressed by introducing isolated cDNA into microorganisms, animal and plant cells, etc. All of this is this enzyme. This enzyme is a novel pyrophosphorylase that has a sequence similar to that of a pyrophosphorylase-specific sequence motif, but forms a population different from that of existing pyrophosphorylases and has a different function.

(Properties of the enzyme protein of the present invention)
This enzyme will be described using an enzyme extracted from pea seedling (Pisum sativum L.) as an example. As a result of molecular weight measurement by SDS-PAGE, the purified enzyme is about 67 kDa. The optimum pH for enzyme activity is 6.5-7.5. Compared to previously known UDP-sugar pyrophosphorylase, which has a higher optimum pH (for example, the optimum pH of potato-derived UDP-glucose pyrophosphorylase is 8.5), the optimum pH of the enzyme Has low features. The enzyme is stable at pH 6.5 to 8.0, and at pH 5.0 or less, the activity drops to 20% or less of the optimum activity. The optimum temperature is 45 ° C, and 99% of its activity is lost at 55 ° C. While this enzyme catalyzes the reaction to produce UDP-sugar and pyrophosphate (PPi) from sugar monophosphate and UTP, it also has the activity of converting UDP-sugar to sugar monophosphate and UTP in the presence of PPi. Have. According to Hanes-Woolf plot, Km value and Vmax of this enzyme are 0.34 mM, 0.96 mM, 0.048 mM, 0.25 Km for glucose monophosphate (Glc 1-P), Ara 1-P, UTP and PPi, respectively. Vmax is 106 μmol / min. / mg protein, 71 μmol / min. / mg protein, 81 μmol / min. / mg protein, and 164 μmol / min. / mg protein, respectively. The enzyme has a wide range of substrate specificities, including L-Ara 1-P, Glc 1-P and galactose monophosphate (Gal 1-P), and Xyl 1-P as a substrate. It reacts slightly with GlcNAc 1-P and does not show activity with glucose 6-phosphate (Glc 6-P). Although this enzyme has high and low activity, it exhibits a wide range of pyrophosphorylase activity against monophosphate of pentose and hexose. This enzyme requires either a metal ion of Mg ²⁺ or Mn ²⁺ for activity expression.
The cDNA of this enzyme is as shown in SEQ ID NO: 4, and its molecular weight is 66,040 kDa (600 amino acids).

(Extraction method)
This enzyme is considered to be widely present in plants, and may be extracted from any plant or from any site. For example, it is contained in pea seedlings (Pisum sativum L.), rice (Oryza sativa), and Arabidopsis thaliana, and can be extracted from these plants. When the distribution of this enzyme was examined in pea seedlings (Pisum sativum L.), it was highest in the upper hypocotyl and lower in the leaf by root, upper hypocotyl, and leaf tissue. On the other hand, the activity per fresh weight was highest in leaves, followed by the hypocotyl and root. It is preferable to extract from the above-ground part including the hypocotyl and the leaf having relatively high activity.

  As long as the activity of the enzyme is not impaired, the extraction may be carried out by any method, and it is preferable to further purify and increase the purity. For example, according to a conventional enzyme purification method, a crude enzyme solution is first prepared, and the enzyme is concentrated by ammonium sulfate fractionation, which can be used for the synthesis of UDP-L-Ara. The plant used for extraction is extracted with a solution that does not impair the enzyme activity, for example, distilled water, a buffer solution, or a salt solution, at 4 ° C. or in ice. Any salt or buffer may be used as long as the pH is 5 or more and 9 or less, but the pH is preferably 6.0 or more and 7.5 or less. Examples of salts and buffers to be used include buffers such as sodium salt, potassium salt, phosphoric acid, Tris-HCl, ethanolamine, acetic acid, and citric acid. The plant body is put into a juicer mixer or the like together with the extraction solvent and crushed, the crushed liquid is filtered, and the supernatant liquid is collected by centrifugation to obtain a crude enzyme solution.

(Concentration / purification method)
Concentration of this enzyme may be carried out by evaporating the water as it is with an evaporator or freeze-drying, or may be precipitated (salted out) with ethanol, ammonium sulfate, sodium sulfate or the like. For example, when ammonium sulfate is added and the enzyme is concentrated, the precipitate when ammonium sulfate is dissolved to 90% saturation in the crude enzyme solution, preferably ammonium sulfate is added to 10% or more to remove the precipitate. It is preferable that the precipitate is recovered by raising the concentration to 80% saturation, more preferably, the precipitate is removed at a saturation concentration of 30% or more, and the precipitate is recovered at a concentration of 75% or less, whereby the enzyme is concentrated.

  Further purification may be performed in order to obtain a highly pure enzyme. For the purification of this enzyme, any method such as dialysis, ultrafiltration, liquid chromatography, electrophoresis, membrane separation, and centrifugation can be used according to a conventional method used for protein purification. For example, in liquid chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, reverse phase chromatography and the like can be carried out alone or in combination. For the purification of this enzyme, a combination of separation by anion exchange chromatography and separation by hydrophobic chromatography is good, and further purity is most preferred by gel filtration chromatography separation. Ion exchange chromatography is preferred because of its increased purity when used repeatedly. In addition, an antibody against the enzyme can be prepared, and the enzyme can be directly purified to high purity by immunochromatography (affinity-chromatography).

(Method for producing UDP-L-Ara pyrophosphorylase)
As described above, this enzyme may be extracted from a plant body, or a protein may be synthesized according to amino acid sequence information. A recombinant protein may be obtained by genetic manipulation using a DNA sequence. Efficiently preferred. For example, the cDNA shown in SEQ ID NO: 4 can be mass-produced by incorporating the cDNA shown in SEQ ID NO: 4 together with an expression vector into a microorganism having established a genetic recombination system such as Escherichia coli, Bacillus subtilis, yeast, and mold. Alternatively, the enzyme can be produced by introducing it into a plant together with a vector that is expressed at a specific site, allowing it to be highly expressed at a specific site, and separating this enzyme. In addition, it can also be produced by incorporating it into insect or animal cells.

(UDP-sugar production method)
Next, a method for producing UDP-sugar using this enzyme will be described. Basically, in addition to this enzyme, a monophosphate corresponding to the target UDP-sugar and UTP are added and reacted under the optimum conditions of the enzyme to produce UDP-sugar. For example, when producing UDP-Glc, in addition to this enzyme, UTP and Glc 1-P are added and reacted to obtain UDP-Glc. When producing UDP-L-Ara, in addition to this enzyme, UTP and L-Ara 1-P are added and reacted to obtain UDP-L-Ara. In addition, UDP-galactose (UDP-Gal), UDP-glucuronic acid (UDP-GlcA), and UDP-xylose (UDP-Xyl) can be obtained in the same manner. Since this enzyme produces UDP-sugars from a variety of sugar monophosphates and UTP, it is possible to produce each UDP-sugar using a single sugar monophosphate as a substrate. Two or more types of UDP-sugars may be synthesized simultaneously by adding more than one type of sugar monophosphate. The synthesis method is specifically pH 4-9, preferably around pH 6-7, and the reaction temperature is 4-60 ° C., preferably 30-45 ° C. under the optimum conditions for the enzyme reaction. In the presence of Mg ²⁺ and Mn ²⁺ necessary for the activity, UTP and sugar monophosphate are added and reacted for several minutes to several hours. Bovine albumin protein or the like may be added to the enzyme reaction solution as an enzyme stabilizer. After completion of the reaction, the enzyme can be inactivated by heating or changing the pH.

  In the presence of PPi, the enzyme of the present invention undergoes the reverse reaction of the reaction of obtaining UDP-sugar from UTP and sugar monophosphate, that is, the reaction of hydrolyzing UDP-sugar to UTP and sugar monophosphate. To do. Therefore, in the synthesis of UDP-sugar, it is desirable to add an enzyme having pyrophosphatase activity, for example, an enzyme derived from yeast to eliminate PPi and suppress the progress of the reverse reaction.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention should not be limited by these examples.

Example 1: (Extraction, isolation and purification of UDP-L-Ara pyrophosphorylase)
A commercially available pea seedling was obtained, held in a growth chamber for 1 day under light irradiation at 25 ° C. and habituated, and then used for enzyme extraction. The crude enzyme was prepared by slightly modifying the method of Kobayashi et al. (Plant Cell Physiol. 43, 1259-1265 (2002)). All operations were performed at 0-4 ° C. 1 kg of pea seedling containing conditioned hypocotyls and leaves is cut finely with a cutter and doubled with extraction buffer (20 mM potassium phosphate (pH 6.9)), 1 mM DTT and 1 mM EDTA It was crushed with a homogenizer. The obtained crushed liquid was filtered through a triple nylon mesh, and 1.8 L of supernatant was obtained by centrifugation (1500 × g, 15 min.) To obtain a crude enzyme solution. Ammonium sulfate was added to the crude enzyme solution until the concentration reached 40%, and a saturated sodium carbonate solution was added to adjust the pH to 6.9. After standing on ice for 30 minutes, the suspension was centrifuged at 12,000 × g for 30 minutes. The supernatant was collected and ammonium sulfate was added to 70%. At this time, the pH of the suspension was adjusted to 6.9. After standing for 30 minutes on ice, the precipitate is collected by centrifugation and suspended in 120 ml of anion exchange chromatography buffer (20 mM potassium phosphate buffer (pH 6.9), 1 mM DTT). Dialyzed overnight against buffer. The obtained ammonium sulfate fraction was stored at 4 ° C. In addition, when UDP-L-Ara pyrophosphorylase activity was measured, the specific activity of the ammonium sulfate fraction was increased 2.8 times compared to the crude enzyme solution.

  Next, the enzyme was purified by chromatographic separation of the obtained ammonium sulfate fraction (40-70% saturated ammonium sulfate precipitation fraction) as follows. The results are shown in FIGS. 1-A, B, C and D.

  First, add approximately 1152 mg of protein in the ammonium sulfate fraction to a DEAE-Sepharose Fast Flow column (2.8 x 73 cm, manufactured by Pharmacia), adsorb it, and buffer solution containing 0 to 0.5 M NaCl (20 mM potassium phosphate buffer). The solution was eluted with a solution (pH 6.9) and 1 mM DTT. The results are shown in FIG. Two fractions with UDP-Glc pyrophosphorylase activity are obtained, and the peak before elution at a NaCl concentration of about 90 mM has UDP-L-Ara pyrophosphorylase activity. It was thought that. The fraction containing the previous peak was designated as an anion exchange column purified fraction. In addition, the specific activity of UDP-L-Ara pyrophosphorylase activity of the fraction purified by anion exchange chromatography was 40 times higher than that of the crude enzyme solution.

  Furthermore, a Butyl-TOYOPEARL 650M column (2 x 19.5 cm: manufactured by Tosoh Corporation) was prepared by adding ammonium sulfate to a purified anion-exchange column fraction (protein amount: 57.71 mg) to 30% and equilibrating with 30% ammonium sulfate solution. ) Was subjected to hydrophobic chromatography (FIG. 1-B). Elution was performed with a linear gradient of ammonium sulfate concentration of 30 to 0% to obtain a single fraction showing enzyme activity, which was purified by hydrophobic chromatography and stored at 4 ° C. In addition, only the result of having monitored the enzyme activity with respect to UDP-Glc pyrophosphorylase activity was shown to FIGS. The UDP-L-Ara pyrophosphorylase activity of the hydrophobic chromatographically purified fraction was 430 times more specific than the crude enzyme solution.

  Ammonium sulfate was added to the hydrophobic chromatographic purified fraction to 80%, and the precipitate was collected by centrifugation. The precipitate (protein amount 1.262 mg) was dissolved in 2 ml of buffer (20 mM potassium phosphate buffer (pH 6.9), 1 mM DTT, 100 mM NaCl), and a Sephacryl S-200 column (2 x 112 cm: Pharmacia) (The result is FIG. 1-C), a fraction showing UDP-Glc pyrophosphorylase activity was obtained, and the fraction was purified by gel filtration chromatography. The UDP-L-Ara pyrophosphorylase activity of the gel filtration chromatography purified fraction was 400 times higher than that of the crude enzyme solution.

  Finally, the gel filtration chromatography purified fraction is again added to the DEAE-Sepharose Fast Flow column (0.7 x 12 cm: Pharmacia), adsorbed, and a buffer solution containing 0 to 0.5 M NaCl (20 mM potassium phosphate buffer solution). (pH 6.9), 1 mM DTT) (FIG. 1-D), a fraction showing UDP-Glc pyrophosphorylase activity was obtained and used as the final column purified fraction. The UDP-L-Ara pyrophosphorylase activity of the final column purified fraction was 1200 times higher in specific activity than the crude enzyme solution, and the yield was 0.4%.

Table 1 summarizes the enzyme activity and yield at each stage where the UDP-L-Ara pyrophosphorylase of the present invention was purified from pea seedlings.

  The final column purified fraction was analyzed by SDS-PAGE. SDS-PAGE was performed according to the method of Laemmli (Leammli, 1970). The acrylamide concentration of the gel was 12%. As molecular weight markers, phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), α-lactalbumin (14 kDa) ) Was used. Protein staining was performed by silver staining. The purified enzyme was observed as a single band, and the molecular weight was calculated to be 67 kDa (see FIG. 2).

Example 2: Synthesis of UDP-sugar using purified enzyme UDP-sugar was synthesized using the purified enzyme obtained in Example 1. When purified enzyme was reacted with 1 mM UTP and Glc 1-P at 25 ° C, UDP-Glc was formed with the reaction time as shown by the black circle in Fig. 3-A. The production amount was about 30% of the addition amount. Further, as shown by the white circle in FIG. 3-A, when the reaction was carried out in the presence of the purified enzyme and PPi, the reverse reaction occurred by PPi, and the amount of UDP-Glc was about 30%. Thus, when pyrophosphatase derived from yeast was added to a system in which the purified enzyme was reacted with 1 mM UTP and Glc 1-P at 25 ° C., PPi was decomposed into phosphate, which is shown in FIG. Thus, 99.7% of the added Glc 1-P was converted to UDP-Glc, and UDP-Glc could be produced efficiently.

In addition, the influence of metal ions on the synthesis was investigated. As a result, as shown in Table 2, the presence of any metal ion of Mg ²⁺ , Mn ²⁺ , Zn ²⁺ is essential for the synthesis of UDP-sugar, and the optimum concentration is 1 to 5 mM. It was. The optimum pH and optimum temperature were pH 6.5 to 7.5 and the optimum temperature was 45 ° C. as shown in FIGS.

  Nucleotide triphosphate (ATP, CTP, GTP, ITP) and PPi were added to the reaction system of 1 mM Glc 1-P and UTP, and purified enzyme, and the influence on the synthesis reaction was examined. Nucleotide triphosphate did not inhibit the synthesis reaction, but was strongly inhibited by PPi.

  Next, the substrate specificity of the purified enzyme was examined. Assuming that the activity of the purified enzyme to Glc 1-P is 100%, the activity to Glc 1-P is 116.2%, the activity to GlcA 1-P is 71.3%, and the activity to L-Ara 1-P 70.5%, activity to Xyl 1-P is 35.9%, activity to GlcNAc 1-P is 4.3%, and the purified enzyme has the ability to act on multiple substrates and synthesize UDP-sugars. It turns out that there is.

  Further, the Km value and Vmax of the purified enzyme are summarized in Table 4. The Km values for UTP and PPi were 0.048 mM and 0.25 mM, which were relatively similar to those derived from humans and potatoes (0.058-0.33 mM and 0.11-2.4 mM). However, the Km values of purified enzymes for Glc 1-P and UDP-Glc are 0.34 mM and 0.34 mM, which are significantly higher than those derived from humans and potatoes (0.14-0.18 mM and 0.04-0.12 mM). The features shown were found. Vmax of purified enzyme is 81 μmol / min. / Mg-protein for UTP, 106 μmol / min. / Mg-protein for glucose monophosphate, and substrate concentration is set to 0.05-0.2 mM It was similar to the Vmax of the potato-derived enzyme determined in 1), ie 94 μmol / min. / Mg-protein for UTP and 64 μmol / min. / Mg-protein for Glc 1-P. However, Vmax for purified enzymes UDP-Glc and PPi is 145 μmol / min. / Mg-protein and 164 μmol / min. / Mg-protein, and 890 μmol / min. / Mg-protein of potato-derived enzyme, The value was low compared to 827 μmol / min. / Mg-protein.

Example 3: Analysis of amino acid sequence After adding 10 μg of the final column purified product obtained in Example 1, V8 protease (manufactured by Wako Pure Chemical Industries, Ltd.) and reacting at 25 ° C. for 15 minutes, SDS-PAGE was performed. Separation and transfer to a PVDF membrane (manufactured by Osmonics) determined the N-terminal sequence. The peptide sequencer used was HPG1000 (manufactured by Hewlett Packard).
The N-terminal sequence of the 31,000 Da polypeptide chain obtained by the enzyme treatment was YNQLDPLxRASGYPD. The 8th x could not be detected. Based on this sequence, DDBJ BLAST search and TAIR BLAST search were performed. The resulting sequence was 86.7% homologous to YNQLDPLLRASGFPD found in the Arabidopsis thaliana open reading frame At5g52560 of unknown function. Moreover, it was 86.7% homologous to YNQLDPLLRASGHPD found in the cDNA sequence AK064009 of rice whose function is not known as in the case of Shironazuna.

Example 4: cDNA cloning and cDNA analysis Materials containing upper axis embryos, leaves, and roots germinated from pea seeds and grown for 7 days were frozen in liquid nitrogen and crushed, and then total RNA was extracted from RNA extraction kit ISOGEN (produced by Nippon Gene Co., Ltd.) ), And a single-stranded cDNA was obtained from reverse transcriptase (ReverTraAce-α: manufactured by Toyobo) and oligo (dT) adapter primer (5′-GCGACATCATCGAATTCCGATGTTTTTTTTTTTTTTT-3 ′).
Using this single-stranded cDNA as a template, degenerate-PCR was performed using two primers, F-1 (5'-CAYGGNCAYGGNGARGT-3 ') and R-1 (5'-GGRTARTCYTGCATCATRCAYTC-3') (condition: denaturation 0.5 min. 94 ° C, annealing 0.5 min. 45 ° C, elongation 1.5 min. 75 ° C, 35 cycles).
The obtained amplified cDNA was subcloned with the pGEM T-Easy vector (Promega) to determine the DNA sequence. ABI310 (Applied Biosystems) was used as the DNA sequencer.

The 3 ′ region of the gene was amplified with two primers (5′-GGCTCACCCACTCTGATGGG-3 ′ and 5′-GCGACATCATCGAATTCCGATG-3 ′) using single-stranded cDNA as a template (conditions: denaturation 0.5 min. 94 ° C., annealing 0.5 min. 55 ° C, elongation 2.0 min. 72 ° C, 30 cycles).
The 5 ′ region of the gene was amplified with two primers (5′-CCATACTTTCAGAATACCGC-3 ′ and 5′-AGAACCCATTTCAACCCGGC-3 ′) using a 5′RACE kit (Invitrogen).
The region encoding this enzyme was amplified using polymerase KOD-plus (manufactured by Toyobo), and the DNA sequence was determined.

The cDNA of this enzyme was as shown in SEQ ID NO: 4, and its molecular weight was 66,040 kDa (600 amino acids).
The 506 bp PCR product obtained from the single-stranded cDNA of pea contained a sequence that matched the sequence obtained from the peptide sequence analysis (YNQLDPLxRASGYPD) (see FIG. 5-A). The full-length cDNA encoding the purified enzyme was obtained by 3'RACE and 5'RACE techniques. The obtained cDNA was designated as PsUSP. PsUSP is a polypeptide chain of 600 amino acids and was calculated to have a molecular weight of 66,040 Da. The peptide sequence obtained from PsUSP did not contain a signal sequence, a mitochondrial targeting sequence, or a chloroplast translocation sequence. Analysis by iPSORT indicated that the enzyme was present in the cytoplasm. The calculated isoelectric point was 5.82. The amino acid sequence deduced from PsUSP was 77% identical to the amino acid sequence obtained from At5g52560 and 72% identical to the amino acid sequence obtained from AK064009. Both potato-derived UDP-Glc pyrophosphorylase and human-derived UDP-GlcNAc pyrophosphorylase were 15% homologous at the amino acid sequence level. In addition, the amino acid sequence deduced from PsUSP contains a region thought to be common to pyrophosphorylases and a uridine binding site (Val-123, Lsy-137, Val-233, Tyr-257). Yes.

  In addition, a comparison between the existing UDP-glucose pyrophosphorylase and UDP-GlcNAc pyrophosphorylase was performed to produce a rootless phylogenetic tree. PsUSP, together with At5g52560 and AK064009, produced UDP-Glc pyrophosphorylase and human Of UDP-GlcNAc pyrophosphorylase was found to belong to a different group (see FIG. 5-B).

Example 5: Expression and purification of recombinant enzyme The coding region was cloned based on the cDNA obtained in Example 4. The coding region was amplified with S-1 (5-GGATCCATGGCTTCCTCCCTCGG-3) and A-1 (5-GAGCTCTCTATATTCTTTGCGCAC-3) containing recognition sites for restriction enzymes BamHI and SacI, and the resulting cDNA fragment was pGEM 5zf + vector ( The DNA sequence was determined by subcloning with Promega. Next, the obtained DNA fragment was introduced into the BamHI and SacI regions of the expression vector pET32a (Novagen), and the recombinant protein was designed to be obtained as a fusion protein of 6xHis-tags and thioredoxin at the N-terminus. Escherichia coli into which the expression vector pET32a incorporating the DNA fragment was introduced was cultured at 10 ° C. and treated with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 24 hours to induce a recombinant fusion protein. After the cells were collected, the cells were lysed with 25 mM potassium phosphate buffer (pH 8.0) containing 0.5 mM phenylmethylsulfonyl fluoride and 0.2% egg white lysozyme. The recombinant fusion protein was washed with a 50 mM sodium phosphate buffer (pH 7.2) containing 50 mM imidazole in addition to a Chelating-SepharoseFF column (Amersham Biosciences), followed by 50 mM phosphate containing 250 mM imidazole. Elute with sodium buffer (pH 7.2). 5 mg purified enzyme was obtained as a fusion protein from 500 ml culture. 0.1 unit of thrombin (Novagen) was added to 1 mg of the fusion protein and reacted at 20 ° C for 24 hours to digest thioredoxin and 6x histidine from the fusion protein, followed by dialysis and purification on a DEAE-SepharoseFF column (0 Elution with -300 mM NaCl) to obtain a purified recombinant enzyme.

  The obtained recombinant enzyme was analyzed by SDS-PAGE. SDS-PAGE was performed in the same manner as the purified enzyme. As a result, a single band was observed, and the molecular weight was calculated to be 70,000 Da (see FIG. 6). Compared to the molecular weight of the natural extract enzyme, 67,000 Da, the molecular weight is large, but this is thought to be derived from the DNA sequence between the PsUSP sequence inserted into the pET32a vector and the thrombin recognition site.

Example 6: Production of UDP-sugars with recombinant enzymes
In a solution containing 50 mM MOPS-KOH buffer (pH 7.0), 5 mM MgCl ₂ , 0.01% BSA, 1 mM L-Ara 1-P, 1 mM UTP, 1 unit / ml pyrophosphatase (derived from yeast) Recombinant enzyme purified to unit / ml was added, and finally 50 ml of the solution was reacted at 35 ° C. for 24 hours. The conversion rate from L-Ara 1-P to UDP-L-Ara was 84% as analyzed by HPLC. After completion of the reaction, it was adsorbed on a charcoal column (4.8 × 10 cm: manufactured by Wako Pure Chemical Industries, Ltd.), and the reaction product was eluted with 70% ethanol containing 0.1 M aqueous ammonia solution. Pepper chromatography using Whatman 3MM filter paper was performed to isolate UDP-L-Ara from the eluate. Salts were removed with a 7: 4: 2 solution in 1-butanol / ethanol / water, then developed with a solution of ethanol / 1 M ammonium acetate (5: 2, pH 6.8), 8 mg of purified UDP-L-Ara Got. The structure of the obtained UDP-L-Ara was confirmed by NMR ( ¹ H- was 600 MHz, ¹³ C- was 150 MHz, ³¹ P- was 243 MHz). ^{In 1} H -NMR (δ = 3.75 ppm) and ¹³ C-NMR (δ = 67.4 ppm), dioxane was used as an internal standard, and in ³¹ P-NMR (δ = 0.0 ppm), phosphoric acid was used as an internal standard. COZY, PFG-HMQC, PFG-HMBC were measured, and the reported NMR spectra of UDP-L-Ara (Pauly M. et al, Anal. Biochem. 278, 69-73, 2000, Ernst C. et al, J. Org. Chem., 68, 5780-5783, 2003). As a result, both agreed and confirmed that the product obtained by the recombinant enzyme was UDP-L-Ara with L-Ara 1-P and UTP as substrates.

Next, the substrate specificity of the recombinant enzyme was examined. The results are shown in Table 5 in comparison with the purified enzyme. Assuming that UDP-Glc pyrophosphorylase activity is 100%, the recombinant enzyme activity is 138.9% for UDP-Gal pyrophosphorylase activity, 62.2% for UDP-GlcA pyrophosphorylase activity, and UDP-Ara The pyrophosphorylase activity was 74.8%, the UDP-Xyl pyrophosphorylase activity was 28.2%, and the UDP-GlcNAc pyrophosphorylase activity was 0.2%.
As with the purified enzyme, the recombinant enzyme was found to have the ability to act on multiple substrates and synthesize UDP-sugars.

Example 7 Production of Arabidopsis At5g52560 Recombinant Protein An Arabidopsis At5g52560 recombinant protein (hereinafter referred to as AT5G52560) was prepared in the same manner as in Examples 4, 5, and 6. At5g52560 was introduced into a pET32a vector, expressed as a fusion protein with a thioredoxin tag (Trx) and a histidine tag (His), and purified with a nickel chelate column (Chelating Sepharose). Thereafter, the tag was removed by digestion with thrombin (Novagen). When the enzyme activity of the At5g52560 recombinant protein was examined, it showed UDP-L-Ara pyrophosphorylase activity similar to that of the purified enzyme. It was also found that the recombinant protein had no effect on its activity even when Trx-His was removed.

Example 8 Production of Rice AK064009 Recombinant Protein A recombinant protein was prepared from a function-unknown gene AK064009 obtained from rice (Oryza sativa) in the same manner as in Examples 4, 5, and 6. When the enzyme activity of the recombinant protein digested with thrombin (Novagen) and without the tag was examined, it showed the same UDP-L-Ara pyrophosphorylase activity as the purified enzyme.

FIG. 1-A is an anion exchange chromatogram of the ammonium sulfate fraction obtained in Example 1, and FIG. 1-B is the hydrophobicity of the purified anion exchange column containing the previous peak of FIG. 1-A. 1-C is a gel filtration chromatogram of the hydrophobic chromatographic purification fraction of FIG. 1-B, and FIG. 1-D is a chromatogram of the gel filtration purification fraction of FIG. 1-C. . FIG. 2 is an SDS-PAGE analysis diagram of the final column purified fraction obtained in Example 1. Fig. 3-A is a graph showing the relationship between the amount of UDP-Glc in the presence of PPi and the reaction time, and Fig. 3-B shows the amount of UDP-Glc and the reaction time when pyrophosphatase (derived from yeast) is added. It is a graph which shows the relationship. FIG. 4-A is a graph showing the relationship between enzyme activity and pH, and FIG. 4-B is a graph showing the relationship between enzyme activity and temperature. FIG. 5-A is an analysis diagram of peptide sequences, and FIG. 5-B is a rootless phylogenetic tree. 6 is an SDS-PAGE analysis diagram of the recombinant enzyme obtained in Example 5. FIG.

Claims (4)

  A polypeptide having a pyrophosphorylase activity comprising the same amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence in which a part of the amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and / or Or enzyme protein. Pea (Pisum sativum L.) or al obtained that according to claim 1, wherein the polypeptide and / or enzyme protein. A polypeptide comprising the same cDNA sequence as shown in SEQ ID NO: 4 or a base sequence in which a part of bases is deleted, substituted or added in the cDNA sequence shown in SEQ ID NO: 4, and comprising the amino acid sequence shown in SEQ ID NO: 1 and / or DNA encoding the polypeptide and / or enzyme protein characterized by having a pyro phosphorylase activity of the enzyme protein.   The same amino acid sequence as any of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, or an amino acid sequence in which some amino acids are deleted, substituted or added in any of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3 A method for producing UDP-sugar, comprising using a polypeptide having pyrophosphorylase activity and / or an enzyme protein.