title: 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.
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 ((2)H/(1)H)(                            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 (2)H associated with this process. The fractionation pattern was investigated in Euphorbia lagascae and Vernonia galamensis, plants that use different enzyme systems to perform the Δ(12)-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 ((2)H/(1)H)(                            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 (2)H isotope profiles, with selection against (2)H in the positions around the Δ(12)-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 ((2)H/(1)H)(                            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 ((2)H/(1)H)(                            i                         ) distribution in the product are the encumbrance of the active site pocket and constraints to conformational readjustment during the linoleate to vernoleate transformation.
authors.affiliation: Elucidation of Biosynthesis by Isotopic Spectrometry Group, CEISAM, University of Nantes-CNRS UMR, France. isabelle.billault@u-psud.fr
authors.forename: Isabelle
authors.initials: I
authors.lastname: Billault
authors.forename: Alicia
authors.initials: A
authors.lastname: Le Du
authors.forename: Minale
authors.initials: M
authors.lastname: Ouethrani
authors.forename: Zeinab
authors.initials: Z
authors.lastname: Serhan
authors.forename: Philippe
authors.initials: P
authors.lastname: Lesot
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Robins
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry*; D028481:Euphorbia / Q000201:enzymology*; D019787:Linoleic Acid / Q000737:chemistry*; D050866:Liquid Crystals / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D009829:Oleic Acids / Q000737:chemistry*; D010084:Oxidation-Reduction; D010938:Plant Oils / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D035741:Vernonia / Q000201:enzymology*
chemical_list: D004852:Epoxy Compounds; D009829:Oleic Acids; D010938:Plant Oils; D010940:Plant Proteins; D012997:Solvents; C081136:vernolic acid; D003577:Cytochrome P-450 Enzyme System; D019787:Linoleic Acid; D003903:Deuterium; D049308:Dioxygenases