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Probing substrate–product relationships by natural abundance deuterium 2D NMR spectroscopy in liquid-crystalline solvents: epoxidation of linoleate to vernoleate by two different plant enzymes

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Abstract

Natural abundance deuterium 2D NMR spectroscopy in weakly ordering, polypeptide chiral liquid crystals is a powerful technique that enables determination of enantiotopic isotopic ratios (2H/1H) i at the methylene groups of long-chain fatty acids. This technique has been used to study the bioconversion of linoleic acid to vernoleic acid with the objective of establishing the in-vivo site-specific fractionation of 2H associated with this process. The fractionation pattern was investigated in Euphorbia lagascae and Vernonia galamensis, plants that use different enzyme systems to perform the Δ12-epoxidation: a cytochrome P450 monooxygenase in the former and a di-iron dioxygenase in the latter. The specific interest in this study was to ascertain whether different (2H/1H) i isotopic ratios in substrate and product might reflect distinct features of the nature of the reaction centre. However, both the linoleate (substrate) samples and both vernoleate (product) samples isolated from the seed oils of the two plants had remarkably similar 2H isotope profiles, with selection against 2H in the positions around the Δ12-epoxidation site. This is interpreted as indicating that, despite differences in the form in which the activated Fe is presented and in the architecture of the active site, the (2H/1H) i isotopic pattern is determined by features common to the reaction. It is suggested that the effects acting as the overall determinants of the final (2H/1H) i distribution in the product are the encumbrance of the active site pocket and constraints to conformational readjustment during the linoleate to vernoleate transformation.

Changes in the (2H/1H)i ratios in converting methyl linoleate to methyl vernoleate in either V. galamensis or E. lagascae indicate isotopic fractionations independent of the type of enzyme reaction centre carrying out the epoxidation

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Acknowledgements

LP and RJR thank the CNRS for regular funding. We thank the United States Department of Agriculture and Professor Sten Stymne for seed oil samples. We are most grateful to Dr Renata Kwiecień for performing the Gaussian09 calculations and to her and Jacques Lebreton for discussions of epoxidation mechanisms.

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  1. Isabelle Billault

    Present address: ICMMO, Université Paris-Sud 11- CNRS UMR8182, 15 rue Georges Clemenceau, 91405, Orsay, France

Authors and Affiliations

  1. Elucidation of Biosynthesis by Isotopic Spectrometry Group, CEISAM, University of Nantes–CNRS UMR6230, BP 92208, 44322, Nantes, France

    Isabelle Billault, Alicia Le Du, Minale Ouethrani & Richard J Robins

  2. RMN en Milieu Orienté, ICMMO, Université de Paris-Sud 11–CNRS UMR8182, 15 rue Georges Clemenceau, 91405, Orsay, France

    Zeinab Serhan & Philippe Lesot

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  1. Isabelle Billault
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Correspondence to Isabelle Billault or Richard J Robins.

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Billault, I., Le Du, A., Ouethrani, M. et al. Probing substrate–product relationships by natural abundance deuterium 2D NMR spectroscopy in liquid-crystalline solvents: epoxidation of linoleate to vernoleate by two different plant enzymes. Anal Bioanal Chem 402, 2985–2998 (2012). https://doi.org/10.1007/s00216-012-5748-6

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