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. 2002 Oct 15;367(Pt 2):491–497. doi: 10.1042/BJ20020103

Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat.

Antonio J Serrato 1, Juan M Pérez-Ruiz 1, Francisco J Cejudo 1
PMCID: PMC1222897  PMID: 12106017

Abstract

Thioredoxins h are ubiquitous proteins reduced by NADPH- thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR-thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat ( Triticum aestivum ). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h ), we successfully reconstituted the wheat NTR-thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.

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Selected References

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  1. Arscott L. D., Gromer S., Schirmer R. H., Becker K., Williams C. H., Jr The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3621–3626. doi: 10.1073/pnas.94.8.3621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Besse I., Wong J. H., Kobrehel K., Buchanan B. B. Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3169–3175. doi: 10.1073/pnas.93.8.3169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buchanan B. B. Regulation of CO2 assimilation in oxygenic photosynthesis: the ferredoxin/thioredoxin system. Perspective on its discovery, present status, and future development. Arch Biochem Biophys. 1991 Jul;288(1):1–9. doi: 10.1016/0003-9861(91)90157-e. [DOI] [PubMed] [Google Scholar]
  4. Dai S., Saarinen M., Ramaswamy S., Meyer Y., Jacquot J. P., Eklund H. Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 A resolution. J Mol Biol. 1996 Dec 20;264(5):1044–1057. doi: 10.1006/jmbi.1996.0695. [DOI] [PubMed] [Google Scholar]
  5. Gautier M. F., Lullien-Pellerin V., de Lamotte-Guéry F., Guirao A., Joudrier P. Characterization of wheat thioredoxin h cDNA and production of an active Triticum aestivum protein in Escherichia coli. Eur J Biochem. 1998 Mar 1;252(2):314–324. doi: 10.1046/j.1432-1327.1998.2520314.x. [DOI] [PubMed] [Google Scholar]
  6. Gladyshev V. N., Jeang K. T., Stadtman T. C. Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6146–6151. doi: 10.1073/pnas.93.12.6146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hirota K., Matsui M., Iwata S., Nishiyama A., Mori K., Yodoi J. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3633–3638. doi: 10.1073/pnas.94.8.3633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Holmgren A., Björnstedt M. Thioredoxin and thioredoxin reductase. Methods Enzymol. 1995;252:199–208. doi: 10.1016/0076-6879(95)52023-6. [DOI] [PubMed] [Google Scholar]
  9. Izawa S., Maeda K., Sugiyama K., Mano J., Inoue Y., Kimura A. Thioredoxin deficiency causes the constitutive activation of Yap1, an AP-1-like transcription factor in Saccharomyces cerevisiae. J Biol Chem. 1999 Oct 1;274(40):28459–28465. doi: 10.1074/jbc.274.40.28459. [DOI] [PubMed] [Google Scholar]
  10. Jacquot J. P., Rivera-Madrid R., Marinho P., Kollarova M., Le Maréchal P., Miginiac-Maslow M., Meyer Y. Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli. J Mol Biol. 1994 Jan 28;235(4):1357–1363. doi: 10.1006/jmbi.1994.1091. [DOI] [PubMed] [Google Scholar]
  11. Kuriyan J., Krishna T. S., Wong L., Guenther B., Pahler A., Williams C. H., Jr, Model P. Convergent evolution of similar function in two structurally divergent enzymes. Nature. 1991 Jul 11;352(6331):172–174. doi: 10.1038/352172a0. [DOI] [PubMed] [Google Scholar]
  12. Laloi C., Rayapuram N., Chartier Y., Grienenberger J. M., Bonnard G., Meyer Y. Identification and characterization of a mitochondrial thioredoxin system in plants. Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):14144–14149. doi: 10.1073/pnas.241340898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lennon B. W., Williams C. H., Jr, Ludwig M. L. Twists in catalysis: alternating conformations of Escherichia coli thioredoxin reductase. Science. 2000 Aug 18;289(5482):1190–1194. doi: 10.1126/science.289.5482.1190. [DOI] [PubMed] [Google Scholar]
  14. Miranda-Vizuete A., Damdimopoulos A. E., Pedrajas J. R., Gustafsson J. A., Spyrou G. Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization. Eur J Biochem. 1999 Apr;261(2):405–412. doi: 10.1046/j.1432-1327.1999.00286.x. [DOI] [PubMed] [Google Scholar]
  15. Mouaheb N., Thomas D., Verdoucq L., Monfort P., Meyer Y. In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3312–3317. doi: 10.1073/pnas.95.6.3312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pedrajas J. R., Kosmidou E., Miranda-Vizuete A., Gustafsson J. A., Wright A. P., Spyrou G. Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae. J Biol Chem. 1999 Mar 5;274(10):6366–6373. doi: 10.1074/jbc.274.10.6366. [DOI] [PubMed] [Google Scholar]
  17. Rivera-Madrid R., Mestres D., Marinho P., Jacquot J. P., Decottignies P., Miginiac-Maslow M., Meyer Y. Evidence for five divergent thioredoxin h sequences in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5620–5624. doi: 10.1073/pnas.92.12.5620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schurmann P., Jacquot J.-P. PLANT THIOREDOXIN SYSTEMS REVISITED. Annu Rev Plant Physiol Plant Mol Biol. 2000 Jun;51(NaN):371–400. doi: 10.1146/annurev.arplant.51.1.371. [DOI] [PubMed] [Google Scholar]
  19. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  20. Serrato A. J., Crespo J. L., Florencio F. J., Cejudo F. J. Characterization of two thioredoxins h with predominant localization in the nucleus of aleurone and scutellum cells of germinating wheat seeds. Plant Mol Biol. 2001 Jun;46(3):361–371. doi: 10.1023/a:1010697331184. [DOI] [PubMed] [Google Scholar]
  21. Spyrou G., Enmark E., Miranda-Vizuete A., Gustafsson J. Cloning and expression of a novel mammalian thioredoxin. J Biol Chem. 1997 Jan 31;272(5):2936–2941. doi: 10.1074/jbc.272.5.2936. [DOI] [PubMed] [Google Scholar]
  22. Tamura T., Stadtman T. C. A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1006–1011. doi: 10.1073/pnas.93.3.1006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Williams C. H., Arscott L. D., Müller S., Lennon B. W., Ludwig M. L., Wang P. F., Veine D. M., Becker K., Schirmer R. H. Thioredoxin reductase two modes of catalysis have evolved. Eur J Biochem. 2000 Oct;267(20):6110–6117. doi: 10.1046/j.1432-1327.2000.01702.x. [DOI] [PubMed] [Google Scholar]
  24. Young T. E., Gallie D. R. Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals. Plant Mol Biol. 1999 Mar;39(5):915–926. doi: 10.1023/a:1006134027834. [DOI] [PubMed] [Google Scholar]

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