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 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.
Cabello-Hurtado
F
F
Département d'Enzymologie Cellulaire et Moléculaire, Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique UPR 406, 28 rue Goethe, 67000 Strasbourg, France.
Batard
Y
Y
Salaün
J P
JP
Durst
F
F
Pinot
F
F
Werck-Reichhart
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry; D005227:Fatty Acids / Q000378:metabolism*; D006368:Helianthus / Q000201:enzymology*; D006801:Humans; D006900:Hydroxylation; D008345:Manganese / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment
D018076:DNA, Complementary; D018744:DNA, Plant; D005227:Fatty Acids; D010940:Plant Proteins; D008345:Manganese; D003577:Cytochrome P-450 Enzyme System; C111492:CYP81B1 protein, Helianthus tuberosus