Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms.
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.
Hughes
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RK
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, U.K. rhughes@bbsrc.ac.uk
Wu
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Robinson
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DS
Hardy
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West
S I
SI
Fairhurst
S A
SA
Casey
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D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D019787:Linoleic Acid / Q000378:metabolism; D015227:Lipid Peroxidation; D008084:Lipoxygenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018532:Pisum sativum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D019787:Linoleic Acid; D008084:Lipoxygenase