CLONING, EXPRESSION IN YEAST, AND FUNCTIONAL-CHARACTERIZATION OF CYP81B1, A PLANT CYTOCHROME-P450 THAT CATALYZES IN-CHAIN HYDROXYLATION OF FATTY-ACIDS

Several omega and in-chain fatty acid hydroxylases have been character ized in higher plants, In microsomes from Helianthus tuberosus tuber t he omega-2, omega-3, and omega-4 hydroxylation of lauric acid is catal yzed by one or a few closely related aminopyrine and MnCl2-inducible c ytochrome 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 expr ession library from aminopyrine-treated tubers led to the identificati on of a cDNA (CYP81B1) corresponding to a transcript induced by aminop yrine, 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), a nd myristic (C14:0) acids, The same metabolites were obtained with tra nsgenic yeast and plant microsomes, a mixture of omega-1 to omega-5 mo nohydroxylated products, The three fatty acids were metabolized with h igh and similar efficiencies, the major position of attack depending o n chain length, When lauric acid was the substrate, turnover was 30.7 +/- 1.4 min(-1) and K-m(app) 788 +/- 400 nM, No metabolism of long cha in fatty acids, aromatic molecules, or herbicides was detected, This n ew fatty acid hydroxylase is typical from higher plants and differs fr om those already isolated from other living organisms.