Plant Molecular Biology

, Volume 33, Issue 2, pp 359–366 | Cite as

Molecular cloning, characterization and expression of cDNA encoding phosphoserine aminotransferase involved in phosphorylated pathway of serine biosynthesis from spinach

  • Kazuki Saito
  • Yoshiko Takagi
  • Ho Chai Ling
  • Hideki Takahashi
  • Masaaki Noji


Phosphoserine aminotransferase (PSA) catalyzes the conversion of phosphohydroxypyruvate to phosphoserine in the phosphorylated pathway of serine biosynthesis. A cDNA clone encoding PSA was isolated from the cDNA library of spinach (Spinacia oleracea L.) green leaves. Determination of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the amino acid sequences of bacterial, yeast and animal PSA. It contains an N-terminal extension of ca. 60 amino acids in addition to the sequences from other organisms. The general features of plastidic transit peptide are observed in this N-terminal sequence, suggesting the plastid localization of the PSA protein encoded by this cDNA. The bacterial expression of the cDNA could functionally rescue the auxotrophy of serine in the serC- mutant, Escherichia coli KL282. The enzymatic activity of PSA was demonstrated in vitro in the extracts of E. coli over-expressing the cDNA. Southern blot analysis indicated the presence of a couple of related genes (Psa) in the spinach genome. RNA blot hybridization suggested the preferential expression of the Psa gene in the roots of green seedlings and in the suspension cells cultured under a dark condition.

cDNA cloning phosphoserine aminotransferase serine biosynthesis spinach Spinacia oleracea 


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  1. 1.
    Belhumeur P, Fortin N, Clark MW: A gene from Saccharomyces cerevisiae which codes for a protein with significant homology to the bacterial 3-phosphoserine aminotransferase. Yeast 10: 385–389 (1994).Google Scholar
  2. 2.
    Boland MJ, Hanks JF, Reynolds PHS, Blevins DG, Tolbert NE, Schubert KR: Subcellular organisation of ureide biogenesis from glycolytic intermediates and ammonia in nitrogen-fixing soybean nodules. Planta 155: 45–51 (1982).Google Scholar
  3. 3.
    Cheung GP, Rosenblum IY, Sallach HJ: Comparative studies of enzymes related to serine metabolism in higher plants. Plant Physiol 43: 1813–1820 (1968).Google Scholar
  4. 4.
    Duncan K, Coggins JR: The serC-aroA peron of Escherichia coli. Biochem J 234: 49–57 (1986).Google Scholar
  5. 5.
    Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb J-F, Dougherty BA, Merrick JM, McKenney K, Sutton G, FitzHugh W, Fields C, Gocayne JD, Scott J, Shirley R, Liu L-I, Glodek A, Kelley JM, Weidman JF, Phillips CA, Spriggs T, Hedblom E, Cotton MD, Utterback RC, Hanna MC, Nguyen DT, Saudek DM, Brandon RC, Fine LD, Frichman JL, Fuhrmann JL, Geoghagen NSM, Gnehm CL, McDonald LA, Small KV, Fraser CM, Smith HO, Venter JC: Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269: 496–512 (1995).Google Scholar
  6. 6.
    Griffin HG: Nucleotide sequence of the Salmonella serC gene. Nucl Acids Res 18: 4260 (1990).Google Scholar
  7. 7.
    Keegstra K, Oslen LJ: Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Physiol Plant Mol Biol 40: 471–501 (1989).Google Scholar
  8. 8.
    Keys AJ: Synthesis and interconversion of Glycine and Serine. In: Miflin BJ (ed) Amino Acids and Derivatives, pp. 359–374. Academic Press, New York (1980).Google Scholar
  9. 9.
    Larsson C, Albertsson E: Enzymes related to serine synthesis in spinach chloroplasts. Physiol Plant 45: 7–10 (1979).Google Scholar
  10. 10.
    Lea PJ, Blackwell RD: The role of amino acid metabolism in photosynthesis. In: Singh BK, Flores HE, Shannon JC (eds) Biosynthesis and Molecular Regulation of Amino Acids in Plants, pp. 98–110. American Society of Plant Physiologists, Rockville (1992).Google Scholar
  11. 11.
    Low B, Gates F, Goldstein T, Soll D: Isolation and partial characterization of temperature-sensitive Escherichia coli mutants with altered leucyl-and seryl-transfer ribonucleic acid synthetases. J Bact 108: 742–750 (1971).Google Scholar
  12. 12.
    Lutcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differs in plants and animals. EMBO J 6: 43–48 (1987).Google Scholar
  13. 13.
    Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).Google Scholar
  14. 14.
    Misrahi M, Atger M, Milgrom E: A novel progesterone-induced messenger RNA in rabbit and human endometria. Cloning and sequence analysis of the complementary DNA. Biochemistry 26: 3975–3982 (1987).Google Scholar
  15. 15.
    Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–479 (1962).Google Scholar
  16. 16.
    Nakagawa N, Tanaka H, Oba T, Ogura N, Ilzuka M: Callus formation from protoplasts of cultured Spinacia oleracea cells. Plant Cell Rep 4: 148–150 (1985).Google Scholar
  17. 17.
    O'Gaora P, Maskell D, Coleman D, Cafferkey M, Dougan G: Cloning and characterization of the serC and aroA genes of Yersinia enterocolitica, and construction of an aroA mutant. Gene 84: 23–30 (1989).Google Scholar
  18. 18.
    Oliver DJ: The glycine decarboxylase multienzyme complex from plant mitochondria. Annu Rev Plant Physiol Plant Mol Biol 45: 323–337 (1994).Google Scholar
  19. 19.
    Ouzounis C, Sander C: Homology of the NifS family of proteins to a new class of pyridoxal phosphate-dependent enzymes. FEBS Lett 322: 159–164 (1993).Google Scholar
  20. 20.
    Reynolds PH, Hine A, Rodber K: Serine metabolism in legume nodules: purification and properties of phosphoserine aminotransferase. Physiol Plant 74: 194–199 (1988).Google Scholar
  21. 21.
    Reynolds PH, Blevis DG: Phosphoserine aminotransferase in soybean root nodules. Plant Physiol 81: 293–293 (1988).Google Scholar
  22. 22.
    Saito K, Tatsuguchi K, Takagi Y, Murakoshi I: Isolation and characterization of cDNA that encodes a putative mitochondrion-localizing isoform of cysteine synthase (Oacetylserine( thiol)-lyase) from Spinacia oleracea. J Biol Chem 269: 28187–28192 (1994).Google Scholar
  23. 23.
    Saito K, Yokoyama H, Noji M, Murakoshi I: Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory roles in cysteine biosynthesis from watermelon. J Biol Chem 270: 16321–16326 (1995).Google Scholar
  24. 24.
    Snell K: The duality of pathways for serine biosynthesis is a fallacy. Trends Biochem Sci 11: 241–243 (1986).Google Scholar
  25. 25.
    Srinivasan R, Oliver DJ: Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex. Plant Physiol 109: 161–168 (1995).Google Scholar
  26. 26.
    Stolz M, Dörnemann D: Purification, characterization and N-terminal sequence of phosphoserine aminotransferase from the green algae Scenedesmus obliquus, mutant C-2 A′. Z Naturforsch 49c: 63–69 (1994).Google Scholar
  27. 27.
    Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase to direct expression cloned genes. Meth Enzymol 185: 60–89 (1990).Google Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Kazuki Saito
    • 1
  • Yoshiko Takagi
    • 1
  • Ho Chai Ling
    • 1
  • Hideki Takahashi
    • 1
  • Masaaki Noji
    • 1
  1. 1.Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology in Research Center of Medicinal ResourcesChiba UniversityChibaJapan

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