CARBOHYDRATES, LIPIDS, AND OTHER NATURAL PRODUCTS
Cloning, Expression in Yeast, and Functional Characterization of CYP81B1, a Plant Cytochrome P450 That Catalyzes In-chain Hydroxylation of Fatty Acids*

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Several ω and in-chain fatty acid hydroxylases have been characterized in higher plants. In microsomes fromHelianthus tuberosus tuber the ω-2, ω-3, and ω-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 ω-1 to ω-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.

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This work was supported in part by the Convention GREG/INRA “Complémentation de la levure par des gènes de plantes.”The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AJ000477 (CYP81B1s) and AJ000478 (CYP81B1l).

These two authors contributed equally to this work.

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Supported by a postdoctoral grant from the Spanish Ministerio de Agricultura, Pesca y Alimentacion.

Supported by the Ministére de la Recherche et de l'Enseignement Supérieur.