Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation

Science

Volumn 280, Issue 5365, 1998, Pages 915-918

  Lee, Michael ^a   Lenman, Marit ^b   Banaś, Antoni ^b   Bafor, Maureen ^b   Singh, Surinder ^c   Schweizer, Michael ^d   Nilsson, Ralf ^e   Liljenberg, Conny ^e   Dahlqvist, Anders ^b   Gummeson, Per Olov ^b   Sjödahl, Staffan ^d   Green, Allan ^c   Stymne, Sten ^b  

^a Svalof Weibull AB   (Sweden)

^b SWEDISH UNIVERSITY OF AGRICULTURAL SCIENCES   (Sweden)

^c CSIRO   (Australia)

^d INSTITUTE OF FOOD RESEARCH   (United Kingdom)

^e UNIVERSITY OF GOTHENBURG   (Sweden)

Author keywords

[No Author keywords available]

Indexed keywords

ENZYME; FATTY ACID;

ARABIDOPSIS; ARTICLE; CATALYSIS; FATTY ACID SYNTHESIS; NONHUMAN; PLANT SEED; PRIORITY JOURNAL; TRANSGENIC PLANT;

ACETYLENE; ALKYNES; AMINO ACID SEQUENCE; ARABIDOPSIS; ASTERACEAE; CATALYSIS; CLONING, MOLECULAR; DNA, COMPLEMENTARY; EPOXY COMPOUNDS; FATTY ACID DESATURASES; GENES, PLANT; IRON; LINOLEIC ACID; MICROSOMES; MOLECULAR SEQUENCE DATA; NAD; NADP; OLEIC ACIDS; OXIDOREDUCTASES; PLANT PROTEINS; PLANTS, GENETICALLY MODIFIED; SACCHAROMYCES CEREVISIAE; SEEDS; SEQUENCE ALIGNMENT;

ARABIDOPSIS; ARABIDOPSIS THALIANA;

EID: 0032496331     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5365.915     Document Type: Article

References (35)

1. F. Bohlman, T. Burkhardt, C. Zdero, Naturally Occurring Acetylenes (Academic Press, New York, 1973).
2. R. C. Badami and K. B. Patil, Prog. Lipid Res. 19, 119 (1981).
3. G. Kohn, E. Hartman, S. Stymne, P. J. Beutelmann, J. Plant Physiol. 144, 265 (1994).
4. W. G. Haigh, L. J. Morris, A. T. James, Lipids 3, 307 (1968).
5. F. R. Earle, A. S. Barclay, I. A. Wolff, ibid. 1, 325 (1966).
6. 14C]18:2-coenzyme A (specific activity 85,000 dpm/nmol) in a total of 360 μl. Preparation of E. lagascae membranes and assay of the epoxyqenase were described in (7) with incubations containing NADPH, unless otherwise indicated in Table 1. Lipid extraction, separation, methylation gas-liquid chromatography (GLC), and radio-GLC were done essentially as in (3, 7).
7. M. Bafor, M. Smith, L. Jonsson, K. Stobart, S. Stymne, Arch. Biochem. Biophys. 303, 145 (1993).
8. 5 was against P-450 reductase that was purified from Helianthus tuberosus.
9. F. J. van der Loo, P. Broun, S. Turner, C. Somerville, Proc. Natl. Acad. Sci. U.S.A 92, 6743 (1995).
10. F. J. van de Loo, B. G. Fox, C. Somerville, in Lipid Metabolism in Plants, T. S. Moore, Ed. (CRC, Boca Raton, FL, 1993), pp. 91-126.
11. 2, were 94°C for 4 min, then 30 cycles of - 94°C for 30 s, 50°C for 30 s, 72°C for 60 s - and finally one cycle of 72°C for 5 min.
12. Cotyledons from seeds harvested 17 to 20 days after flowering, were used to make a cDNA library with a Uni-ZAP XR cDNA cloning kit from Stratagene. After screening with a random primed probe, pBluescript phagemid was excised. This was used to create a double-stranded DNA plasmid.
13. Plasmid pVT-Crep1 was constructed by placing the insert from pCrep1 into the vector pVT100U, which contains the constitutive alcohol dehydrogenase promoter [T. Vernet, D. Dignard, D. Y. Thomas, Gene 52, 225 (1987); R. Elble, Biotechniques 13, 18 (1992)].
14. Plasmid pVT-Crep1 was constructed by placing the insert from pCrep1 into the vector pVT100U, which contains the constitutive alcohol dehydrogenase promoter [T. Vernet, D. Dignard, D. Y. Thomas, Gene 52, 225 (1987); R. Elble, Biotechniques 13, 18 (1992)].
15. Yeast were grown in liquid medium without uracil at 28°C for 5 hours before linoleic acid and Tween 40 were added to a final concentration of 0.03% (w/v) and 1% (w/v), respectively. After a further 78 hours of cultivation, cells were washed and the lipids extracted. Methyl esters were prepared with 4% w/w methanolic HCI and analyzed by GLC with a glass column (2.5m long with a 2-mm inner diameter) packed with 3% SP-2300 on Supelcoport 100/120 mesh (Supelco, Bellefonte, PA).
16. FADEA was prepared [R. Nilsson and C. Liljenberg, Phytochem. Anal. 2, 253 (1991)] and injected directly for GLC-MS [Hewlett-Packard 5890 II GLC with a DB225 (J & W Scientific, Folsom, CA) in series with a Rtx 2330 (Restek, Bellefonte, PA) fused-silica capillary column coupled to a Hewlett-Packard 5989A mass spectrometer working in electron impact mode at 70 eV].
17. A binary vector for the acetylenase consisted of the Crep1 cDNA placed downstream of the -309 fragment from the napin promoter [K. Stålberg, M. Ellerström, L. Josefsson, L. Rask, Plant Mol. Biol. 23, 671 (1993)] in the vector pGPTV-KAN [D. Becker, E. Kemper, J. Schell, R. Masterson, ibid. 20, 1195 (1992)]. Arabidopsis thaliana Columbia (C-24) was transformed with Agrobacterium tumefaciens [D. Valvekens, M. Van Montagu, Van Lusbettens, Proc. Natl. Acad. Sci. U.S.A. 85, 5536 (1988)].
18. A binary vector for the acetylenase consisted of the Crep1 cDNA placed downstream of the -309 fragment from the napin promoter [K. Stålberg, M. Ellerström, L. Josefsson, L. Rask, Plant Mol. Biol. 23, 671 (1993)] in the vector pGPTV-KAN [D. Becker, E. Kemper, J. Schell, R. Masterson, ibid. 20, 1195 (1992)]. Arabidopsis thaliana Columbia (C-24) was transformed with Agrobacterium tumefaciens [D. Valvekens, M. Van Montagu, Van Lusbettens, Proc. Natl. Acad. Sci. U.S.A. 85, 5536 (1988)].
19. A binary vector for the acetylenase consisted of the Crep1 cDNA placed downstream of the -309 fragment from the napin promoter [K. Stålberg, M. Ellerström, L. Josefsson, L. Rask, Plant Mol. Biol. 23, 671 (1993)] in the vector pGPTV-KAN [D. Becker, E. Kemper, J. Schell, R. Masterson, ibid. 20, 1195 (1992)]. Arabidopsis thaliana Columbia (C-24) was transformed with Agrobacterium tumefaciens [D. Valvekens, M. Van Montagu, Van Lusbettens, Proc. Natl. Acad. Sci. U.S.A. 85, 5536 (1988)].
20. Methyl esters were prepared by heating 10 to 30 whole seeds at 85°C for 90 min in 1 ml of 0.1 M sodium methoxide. Methyl esters were extracted with hexane and analyzed by GLC through a 50 m by 0.32 mm CP-Wax58-CB fused-silica column (Chrompack).
21. A C. palaestina library was prepared and screened as described (12).
22. The Cpal2 transformation was done as described (76) except that the binary vector used was pBI121 (Clonetech).
23. 18 fatty acid.
24. N. Engeseth and S. Stymne, Planta 198, 238 (1996).
25. P. Broun, N. Hawker, C. R. Somerville, in Physiology, Biochemistry and Molecular Biology of Plant Lipids, J. P. Williams, U. K. Mobashsher, N. W. Lem, Eds. (Kluwer Academic, Dordrecht, Netherlands 1996), pp. 342-344.
26. J. Shanklin, E. Whittle, B. G. Fox, Biochemistry 33, 12787 (1994).
27. J. Shanklin et al., in (22), pp. 6-11.
28. J. Okuley et al., Plant Cell 6, 147 (1994).
29. J. Shanklin, J. Achim, H. Schmidt, B. G. Fox, E. Munck, Proc. Natl. Acad. Sci. U.S.A. 94, 2981 (1997).
30. J. E. Colbert, A. G. Katapodis, S. W. May, J. Am. Chem. Soc. 112, 3993 (1990).
31. L. Shu et al., Science 275, 515 (1997).
32. M. O. Bagby, M. Mikolajczak, K. L. Mikolajczak, Alkali Isomerization of Crepenynic Acid to 8,10,12-Octadecatrienoic Acid. U.S. patent no. 3,356,699 (1967).
33. European Molecular Biology Laboratory (EMBL) accession numbers are as follows: C. palaestina epoxygenase, Y16283; C. alpina acetylenase, Y16285; C. palaestina putative Δ12 desaturase, Y16284; A thaliana Δ12 desaturase, L26296; and the castor bean oleate hydroxylase, U22378.
34. Pileup and Pretty, Wisconsin Package Version 9.0-UNIX, Genetics Computer Group, Madison, WI.
35. 5 to P-450 reductase and S. McKinney and A. T. Carter for technical assistance. Funded in part by the Swedish Foundation for Agricultural Research, the Swedish Natural Science Research Council, Stiftelsen Svensk Oljeväxtforskning, and European Union Grant number AIR2-CT94-0967.