Cloning and functional characterization of CYP94A2, a medium chain fatty acid hydroxylase from Vicia sativa.
A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A2) was isolated from a Vicia sativa library. CYP94A2 displays 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolated from the same material. Heterologous expression of CYP94A2 in Saccharomyces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K(m(app)) of 4.0 microM and a turnover rate number of 80 min(-1). In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidized. Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain. Northern blot analysis showed a low level of constitutive expression of CYP94A2 in V. sativa seedlings. In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings treated with clofibrate or methyl jasmonate, indicating that both substrate range and gene regulation of the two fatty acid hydroxylases are different.
Département d'Enzymologie Cellulaire et Moléculaire, Institut de Biologie Moléculaire des Plantes-CNRS UPR 406, 28 rue Goethe, Strasbourg Cedex, F-67083, France.
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Le Bouquin
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Pinot
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Benveniste
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JP
Salaün
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Durst
D000085:Acetates / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002994:Clofibrate / Q000494:pharmacology; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D007887:Fabaceae / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019814:Myristic Acid / Q000378:metabolism; D054883:Oxylipins; D010946:Plants, Medicinal; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012639:Seeds / Q000187:drug effects / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000085:Acetates; D003517:Cyclopentanes; D054883:Oxylipins; D019814:Myristic Acid; C072239:methyl jasmonate; D006899:Mixed Function Oxygenases; C019081:fatty acid alpha-hydroxylase; D002994:Clofibrate