Abstract
Several omega and in-chain fatty acid hydroxylases have been characterized in higher plants. In microsomes from Helianthus tuberosus tuber the omega-2, omega-3, and omega-4 hydroxylation of lauric acid is catalyzed by one or a few closely related aminopyrine- and MnCl2-inducible cytochrome P450(s). To isolate the cDNA and determine the sequences of the(se) enzyme(s), we used antibodies directed against a P450-enriched fraction purified from Mn2+-induced tissues. Screening of a cDNA expression library from aminopyrine-treated tubers led to the identification of a cDNA (CYP81B1) corresponding to a transcript induced by aminopyrine. CYP81B1 was expressed in yeast. A systematic exploration of its function revealed that it specifically catalyzes the hydroxylation of medium chain saturated fatty acids, capric (C10:0), lauric (C12:0), and myristic (C14:0) acids. The same metabolites were obtained with transgenic yeast and plant microsomes, a mixture of omega-1 to omega-5 monohydroxylated products. The three fatty acids were metabolized with high and similar efficiencies, the major position of attack depending on chain length. When lauric acid was the substrate, turnover was 30.7 +/- 1.4 min-1 and Km(app) 788 +/- 400 nM. No metabolism of long chain fatty acids, aromatic molecules, or herbicides was detected. This new fatty acid hydroxylase is typical from higher plants and differs from those already isolated from other living organisms.
Highlights
Engineering of lipid metabolism in oilseed crops has become one of the major objectives of plant biotechnology [1,2,3,4]
Isolation of the CYP81B1 cDNA—We previously raised antibodies against a P450-enriched fraction partially purified from MnCl2-induced H. tuberosus tuber tissues [27]
Small increases in activity are observed after mechanical stress, such as wounding or aging of the shoots or tissues, but these increases are negligible compared with the response to some specific chemical treatments [15]
Summary
Engineering of lipid metabolism in oilseed crops has become one of the major objectives of plant biotechnology [1,2,3,4]. Attack of medium chain fatty acids, capric (C10:0), lauric (C12:0), and myristic acids (C14:0), by P450 enzymes occurs at different positions, depending on plant species. A second type of in-chain metabolism was first described in Helianthus tuberosus tuber [21,22,23], but is observed in several monocots like maize, tulip, or lily [17] It results in the formation of -2, -3, and -4 monohydroxylated products. This type of fatty acid metabolism is strongly induced in response to MnCl2, aminopyrine, and phenobarbital treatments of plant tissues [17, 22, 24]. This paper is available on line at http://www.jbc.org involvement of several and probably closely related enzymes [23, 25, 26]
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