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J. Biol. Chem., Vol. 280, Issue 18, 17645-17651, May 6, 2005

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Quantitative Deuterium Isotopic Profiling at Natural Abundance Indicates Mechanistic Differences for ¹²-Epoxidase and ¹²-Desaturase in Vernonia galamensis^*

Isabelle Billault, Jia-Rong Duan, Sébastien Guiet, and Richard J. Robins

From the Groupe de Fractionnement Isotopique de Métabolismes, Laboratoire d'Analyse Isotopique et Electrochimique de Métabolismes, CNRS UMR6006, Université de Nantes, BP 99208, F-44322 Nantes, France

Quantitative ²H NMR spectroscopy can determine the natural abundance (²H/¹H) ratio at each site of a molecule. In natural products, variation in these values is related to the reaction mechanisms in the pertinent biosynthetic pathway. For the first time, this novel approach has been exploited to probe for mechanistic differences in the introduction of different functionalities into a long-chain fatty acid. Vernolic acid, a major component of the seed oil of Vernonia galamensis, contains both an epoxide and a desaturation. The site-specific isotopic distribution (²H/¹H)_i has been determined for both vernolic acid and linoleic acid isolated from the same V. galamensis oil. It is found that the (²H/¹H) ratio of vernolic acid shows a pattern along the entire length of the chain, consistent with linoleic acid being its immediate precursor. Notably, the C13 relates to the C13 of linoleic acid but not to the C13 of oleic acid. Furthermore, the C12 and C13 positions in vernolic acid are less depleted, consistent with a change in hybridization state from sp² to sp³. However, the C11 position shows a marked relative enrichment in the vernolic acid, implying that it plays a role in the epoxidase but not the desaturase mechanism. Thus, although it can be concluded that the catalytic mechanisms for the epoxidase and desaturase activities are similar, marked differences in the residual (²H/¹H) patterns indicate that the reaction mechanisms are not identical.

Received for publication, January 25, 2005 , and in revised form, February 17, 2005.

^* This work was supported by Eurofins Scientific Analytic (to S. G.) and the French Ministry of Foreign Affairs (to J.-R. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: LAIEM, CNRS UMR6006, University of Nantes, BP 99208, F-44322 Nantes, France. Tel.: 332-51-12-57-07; Fax: 332-51-12-57-12; E-mail: isabelle.billault{at}univ-nantes.fr.

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