Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Jul 1;333(Pt 1):33–43. doi: 10.1042/bj3330033

Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms.

R K Hughes 1, Z Wu 1, D S Robinson 1, D Hardy 1, S I West 1, S A Fairhurst 1, R Casey 1
PMCID: PMC1219552  PMID: 9639559

Abstract

The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L. cv. Birte) seeds have been cloned and expressed from full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison of both isoforms purified to apparent homogeneity from E. coli and pea seeds has confirmed the authenticity of the recombinant products and established the properties of the native enzymes. Despite 86% similarity at the amino acid sequence level, the enzymes have distinct properties. They have been characterized in terms of specific activity, Fe content, optimum pH, substrate and product specificity, apparent Km and Vmax for the preferred substrate, linoleic acid, and interfacial behaviour with linoleic acid. We have used this evidence, in addition to EPR spectroscopy of the hydroperoxide-activated enzymes and estimates of kcat/Km, to propose different reaction mechanisms for linoleic acid oxidation for the two isoforms. The differences relate primarily to carbonyl production from linoleic acid for which we propose a mechanism. This implicates the release of a peroxyl radical in an aerobic hydroperoxidase reaction, as the source of the carbonyl compounds formed by dismutation of the liberated peroxyl radical.

Full Text

The Full Text of this article is available as a PDF (669.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Bulen W. A., LeComte J. R. The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci U S A. 1966 Sep;56(3):979–986. doi: 10.1073/pnas.56.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chasteen N. D., Grady J. K., Skorey K. I., Neden K. J., Riendeau D., Percival M. D. Characterization of the non-heme iron center of human 5-lipoxygenase by electron paramagnetic resonance, fluorescence, and ultraviolet-visible spectroscopy: redox cycling between ferrous and ferric states. Biochemistry. 1993 Sep 21;32(37):9763–9771. doi: 10.1021/bi00088a031. [DOI] [PubMed] [Google Scholar]
  4. Donnelly J. K., Robinson D. S. Free radicals in foods. Free Radic Res. 1995 Feb;22(2):147–176. doi: 10.3109/10715769509147536. [DOI] [PubMed] [Google Scholar]
  5. Ealing P. M., Casey R. The cDNA cloning of a pea (Pisum sativum) seed lipoxygenase. Sequence comparisons of the two major pea seed lipoxygenase isoforms. Biochem J. 1989 Dec 15;264(3):929–932. doi: 10.1042/bj2640929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ealing P. M., Casey R. The complete amino acid sequence of a pea (Pisum sativum) seed lipoxygenase predicted from a near full-length cDNA. Biochem J. 1988 Aug 1;253(3):915–918. doi: 10.1042/bj2530915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forster C., Knox M., Domoney C., Casey R. lox1:Ps:2, a Pisum sativum seed lipoxygenase gene. Plant Physiol. 1994 Nov;106(3):1227–1228. doi: 10.1104/pp.106.3.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gaffney B. J. Lipoxygenases: structural principles and spectroscopy. Annu Rev Biophys Biomol Struct. 1996;25:431–459. doi: 10.1146/annurev.bb.25.060196.002243. [DOI] [PubMed] [Google Scholar]
  9. Gardner H. W. Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism. Biochim Biophys Acta. 1989 Feb 20;1001(3):274–281. doi: 10.1016/0005-2760(89)90111-2. [DOI] [PubMed] [Google Scholar]
  10. Garssen G. J., Vliegenthart J. F., Boldingh J. An anaerobic reaction between lipoxygenase, linoleic acid and its hydroperoxides. Biochem J. 1971 Apr;122(3):327–332. doi: 10.1042/bj1220327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Glickman M. H., Klinman J. P. Lipoxygenase reaction mechanism: demonstration that hydrogen abstraction from substrate precedes dioxygen binding during catalytic turnover. Biochemistry. 1996 Oct 1;35(39):12882–12892. doi: 10.1021/bi960985q. [DOI] [PubMed] [Google Scholar]
  12. Kanofsky J. R., Axelrod B. Singlet oxygen production by soybean lipoxygenase isozymes. J Biol Chem. 1986 Jan 25;261(3):1099–1104. [PubMed] [Google Scholar]
  13. Kramer J. A., Johnson K. R., Dunham W. R., Sands R. H., Funk M. O., Jr Position 713 is critical for catalysis but not iron binding in soybean lipoxygenase 3. Biochemistry. 1994 Dec 20;33(50):15017–15022. doi: 10.1021/bi00254a010. [DOI] [PubMed] [Google Scholar]
  14. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  15. Kühn H., Wiesner R., Rathmann J., Schewe T. Formation of ketodienoic fatty acids by the pure pea lipoxygenase-1. Eicosanoids. 1991;4(1):9–14. [PubMed] [Google Scholar]
  16. 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]
  17. Lu K. V., Rohde M. F., Thomason A. R., Kenney W. C., Lu H. S. Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli. Biochem J. 1995 Jul 15;309(Pt 2):411–417. doi: 10.1042/bj3090411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ludwig P., Holzhütter H. G., Colosimo A., Silvestrini M. C., Schewe T., Rapoport S. M. A kinetic model for lipoxygenases based on experimental data with the lipoxygenase of reticulocytes. Eur J Biochem. 1987 Oct 15;168(2):325–337. doi: 10.1111/j.1432-1033.1987.tb13424.x. [DOI] [PubMed] [Google Scholar]
  19. Minor W., Steczko J., Stec B., Otwinowski Z., Bolin J. T., Walter R., Axelrod B. Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution. Biochemistry. 1996 Aug 20;35(33):10687–10701. doi: 10.1021/bi960576u. [DOI] [PubMed] [Google Scholar]
  20. Peng Y. L., Shirano Y., Ohta H., Hibino T., Tanaka K., Shibata D. A novel lipoxygenase from rice. Primary structure and specific expression upon incompatible infection with rice blast fungus. J Biol Chem. 1994 Feb 4;269(5):3755–3761. [PubMed] [Google Scholar]
  21. Prigge S. T., Boyington J. C., Gaffney B. J., Amzel L. M. Structure conservation in lipoxygenases: structural analysis of soybean lipoxygenase-1 and modeling of human lipoxygenases. Proteins. 1996 Mar;24(3):275–291. doi: 10.1002/(SICI)1097-0134(199603)24:3<275::AID-PROT1>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  22. Regdel D., Schewe T., Rapoport S. M. Enzymatic properties of the lipoxygenase from pea seeds. Biomed Biochim Acta. 1985;44(10):1411–1428. [PubMed] [Google Scholar]
  23. Royo J., Vancanneyt G., Pérez A. G., Sanz C., Störmann K., Rosahl S., Sánchez-Serrano J. J. Characterization of three potato lipoxygenases with distinct enzymatic activities and different organ-specific and wound-regulated expression patterns. J Biol Chem. 1996 Aug 30;271(35):21012–21019. doi: 10.1074/jbc.271.35.21012. [DOI] [PubMed] [Google Scholar]
  24. Schewe T., Rapoport S. M., Kühn H. Enzymology and physiology of reticulocyte lipoxygenase: comparison with other lipoxygenases. Adv Enzymol Relat Areas Mol Biol. 1986;58:191–272. doi: 10.1002/9780470123041.ch6. [DOI] [PubMed] [Google Scholar]
  25. Schilstra M. J., Veldink G. A., Vliegenthart J. F. Effect of nonionic detergents on lipoxygenase catalysis. Lipids. 1994 Apr;29(4):225–231. doi: 10.1007/BF02536325. [DOI] [PubMed] [Google Scholar]
  26. Schilstra M. J., Veldink G. A., Vliegenthart J. F. Kinetic analysis of the induction period in lipoxygenase catalysis. Biochemistry. 1993 Aug 3;32(30):7686–7691. doi: 10.1021/bi00081a012. [DOI] [PubMed] [Google Scholar]
  27. Shibata D., Axelrod B. Plant lipoxygenases. J Lipid Mediat Cell Signal. 1995 Oct;12(2-3):213–228. doi: 10.1016/0929-7855(95)00020-q. [DOI] [PubMed] [Google Scholar]
  28. Shirano Y., Shibata D. Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level. FEBS Lett. 1990 Oct 1;271(1-2):128–130. doi: 10.1016/0014-5793(90)80388-y. [DOI] [PubMed] [Google Scholar]
  29. Skrzypczak-Jankun E., Amzel L. M., Kroa B. A., Funk M. O., Jr Structure of soybean lipoxygenase L3 and a comparison with its L1 isoenzyme. Proteins. 1997 Sep;29(1):15–31. [PubMed] [Google Scholar]
  30. Slappendel S., Veldink G. A., Vliegenthart J. F., Aasa R., Malmström B. G. EPR spectroscopy of soybean lipoxygenase-1. Description and quantification of the high-spin fe(III) signals. Biochim Biophys Acta. 1981 Jan 30;667(1):77–86. doi: 10.1016/0005-2795(81)90068-4. [DOI] [PubMed] [Google Scholar]
  31. Sloane D. L., Leung R., Craik C. S., Sigal E. A primary determinant for lipoxygenase positional specificity. Nature. 1991 Nov 14;354(6349):149–152. doi: 10.1038/354149a0. [DOI] [PubMed] [Google Scholar]
  32. Steczko J., Donoho G. A., Dixon J. E., Sugimoto T., Axelrod B. Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli. Protein Expr Purif. 1991 Apr-Jun;2(2-3):221–227. doi: 10.1016/1046-5928(91)90075-t. [DOI] [PubMed] [Google Scholar]
  33. Studier F. W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991 May 5;219(1):37–44. doi: 10.1016/0022-2836(91)90855-z. [DOI] [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Verhagen J., Vliegenthart J. F., Boldingh J. Micelle and acid-soap formation of linoleic acid and 13-L-hydroperoxylinoleic acid being substrates of lipoxygenase-1. Chem Phys Lipids. 1978 Nov;22(4):255–259. doi: 10.1016/0009-3084(78)90014-2. [DOI] [PubMed] [Google Scholar]
  36. Wada K., Wada Y., Doi H., Ishibashi F., Gojobori T., Ikemura T. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):1981–1986. doi: 10.1093/nar/19.suppl.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. de Groot J. J., Veldink G. A., Vliegenthart J. F., Boldingh J., Wever R., van Gelder B. F. Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1. Biochim Biophys Acta. 1975 Jan 23;377(1):71–79. doi: 10.1016/0005-2744(75)90287-9. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES