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. 1997 Aug;114(4):1169–1175. doi: 10.1104/pp.114.4.1169

High-yield expression of pea thioredoxin m and assessment of its efficiency in chloroplast fructose-1,6-bisphosphatase activation.

J López Jaramillo 1, A Chueca 1, J P Jacquot 1, R Hermoso 1, J J Lázaro 1, M Sahrawy 1, J López Gorgé 1
PMCID: PMC158409  PMID: 9276945

Abstract

A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L. Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/ FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schürmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37-45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.

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Selected References

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  1. Bassham J. A., Krause G. H. Free energy changes and metabolic regulation in steady-state photosynthetic carbon reduction. Biochim Biophys Acta. 1969 Oct 21;189(2):207–221. doi: 10.1016/0005-2728(69)90048-6. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brandes H. K., Larimer F. W., Geck M. K., Stringer C. D., Schürmann P., Hartman F. C. Direct identification of the primary nucleophile of thioredoxin f. J Biol Chem. 1993 Sep 5;268(25):18411–18414. [PubMed] [Google Scholar]
  4. Carrasco J. L., Chueca A., Prado F. E., Hermoso R., Lázaro J. J., Ramos J. L., Sahrawy M., López Gorgé J. Cloning, structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme. Planta. 1994;193(4):494–501. doi: 10.1007/BF02411553. [DOI] [PubMed] [Google Scholar]
  5. Crawford N. A., Yee B. C., Hutcheson S. W., Wolosiuk R. A., Buchanan B. B. Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants. Arch Biochem Biophys. 1986 Jan;244(1):1–15. doi: 10.1016/0003-9861(86)90088-3. [DOI] [PubMed] [Google Scholar]
  6. Eklund H., Gleason F. K., Holmgren A. Structural and functional relations among thioredoxins of different species. Proteins. 1991;11(1):13–28. doi: 10.1002/prot.340110103. [DOI] [PubMed] [Google Scholar]
  7. Geck M. K., Larimer F. W., Hartman F. C. Identification of residues of spinach thioredoxin f that influence interactions with target enzymes. J Biol Chem. 1996 Oct 4;271(40):24736–24740. doi: 10.1074/jbc.271.40.24736. [DOI] [PubMed] [Google Scholar]
  8. Hermoso R., Castillo M., Chueca A., Lázaro J. J., Sahrawy M., Gorgé J. L. Binding site on pea chloroplast fructose-1,6-bisphosphatase involved in the interaction with thioredoxin. Plant Mol Biol. 1996 Feb;30(3):455–465. doi: 10.1007/BF00049324. [DOI] [PubMed] [Google Scholar]
  9. Hirel P. H., Schmitter M. J., Dessen P., Fayat G., Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huppe H. C., Buchanan B. B. Activation of a chloroplast type of fructose bisphosphatase from Chlamydomonas reinhardtii by light-mediated agents. Z Naturforsch C. 1989 May-Jun;44(5-6):487–494. doi: 10.1515/znc-1989-5-624. [DOI] [PubMed] [Google Scholar]
  11. Häberlein I., Vogeler B. Completion of the thioredoxin reaction mechanism: kinetic evidence for protein complexes between thioredoxin and fructose 1,6-bisphosphatase. Biochim Biophys Acta. 1995 Dec 6;1253(2):169–174. doi: 10.1016/0167-4838(95)00153-1. [DOI] [PubMed] [Google Scholar]
  12. Hög J. O., von Bahr-Lindström H., Josephson S., Wallace B. J., Kushner S. R., Jörnvall H., Holmgren A. Nucleotide sequence of the thioredoxin gene from Escherichia coli. Biosci Rep. 1984 Nov;4(11):917–923. doi: 10.1007/BF01116889. [DOI] [PubMed] [Google Scholar]
  13. Kamo M., Tsugita A., Wiessner C., Wedel N., Bartling D., Herrmann R. G., Aguilar F., Gardet-Salvi L., Schürmann P. Primary structure of spinach-chloroplast thioredoxin f. Protein sequencing and analysis of complete cDNA clones for spinach-chloroplast thioredoxin f. Eur J Biochem. 1989 Jun 15;182(2):315–322. doi: 10.1111/j.1432-1033.1989.tb14832.x. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lepiniec L., Hodges M., Gadal P., Crétin C. Isolation, characterization and nucleotide sequence of a full-length pea cDNA encoding thioredoxin-f. Plant Mol Biol. 1992 Mar;18(5):1023–1025. doi: 10.1007/BF00019224. [DOI] [PubMed] [Google Scholar]
  16. López Jaramillo J., Chueca A., Sahrawy M., Hermoso R., Lázaro J. J., Prado F. E., López Gorgé J. Cloning and sequencing of a pea cDNA fragment coding for thioredoxin m. Plant Physiol. 1994 Jul;105(3):1021–1022. doi: 10.1104/pp.105.3.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maeda K., Tsugita A., Dalzoppo D., Vilbois F., Schürmann P. Further characterization and amino acid sequence of m-type thioredoxins from spinach chloroplasts. Eur J Biochem. 1986 Jan 2;154(1):197–203. doi: 10.1111/j.1432-1033.1986.tb09379.x. [DOI] [PubMed] [Google Scholar]
  18. Pla A., Lopez-Gorge J. Thioredoxin/fructose-1,6-bisphosphatase affinity in the enzyme activation by the ferredoxin-thioredoxin system. Biochim Biophys Acta. 1981 Jun 12;636(1):113–118. doi: 10.1016/0005-2728(81)90082-7. [DOI] [PubMed] [Google Scholar]
  19. Salamon Z., Tollin G., Hirasawa M., Gardet-Salvi L., Stritt-Etter A. L., Knaff D. B., Schürmann P. The oxidation-reduction properties of spinach thioredoxins f and m and of ferredoxin:thioredoxin reductase. Biochim Biophys Acta. 1995 Jun 30;1230(3):114–118. doi: 10.1016/0005-2728(95)00042-h. [DOI] [PubMed] [Google Scholar]
  20. Schürmann P., Maeda K., Tsugita A. Isomers in thioredoxins of spinach chloroplasts. Eur J Biochem. 1981 May;116(1):37–45. doi: 10.1111/j.1432-1033.1981.tb05297.x. [DOI] [PubMed] [Google Scholar]
  21. Stein M., Jacquot J. P., Jeannette E., Decottignies P., Hodges M., Lancelin J. M., Mittard V., Schmitter J. M., Miginiac-Maslow M. Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties. Plant Mol Biol. 1995 Jun;28(3):487–503. doi: 10.1007/BF00020396. [DOI] [PubMed] [Google Scholar]
  22. Wedel N., Clausmeyer S., Herrmann R. G., Gardet-Salvi L., Schürmann P. Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts. Plant Mol Biol. 1992 Feb;18(3):527–533. doi: 10.1007/BF00040668. [DOI] [PubMed] [Google Scholar]
  23. Wolosiuk R. A., Crawford N. A., Yee B. C., Buchanan B. B. Isolation of three thioredoxins from spinach leaves. J Biol Chem. 1979 Mar 10;254(5):1627–1632. [PubMed] [Google Scholar]
  24. de Lamotte-Guery F., Miginiac-Maslow M., Decottignies P., Stein M., Minard P., Jacquot J. P. Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes. Eur J Biochem. 1991 Mar 14;196(2):287–294. doi: 10.1111/j.1432-1033.1991.tb15816.x. [DOI] [PubMed] [Google Scholar]

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