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The origin of hydrogen atoms during fatty acid biosynthesis in Fusarium lateritium has been quantified by isotope tracking close to natural abundance. Methyl linoleate was isolated from F. lateritium grown in natural abundance medium or in medium slightly enriched with labeled water, glucose, or acetate, and the 2 H incorporation was determined by quantitative 2 H-{ 1 H} NMR in isotropic and chiral oriented solvents. Thus, the individual ( 2 H/ 1 H) i ratio at each pro-R and pro-S hydrogen position of the CH 2 groups along the chain can be analyzed. These values allow the isotope redistribution coefficients (a ij ) that characterize the specific source of each hydrogen atom to be related to the nonexchangeable hydrogen atoms in glucose and to the medium water. In turn, these can be related to the stereoselectivity that operates during the introduction or removal of hydrogens along the fatty acid chain. First, at even CH 2 the pro-S hydrogen comes only from water by protonation, whereas the pro-R hydrogen is introduced partly via acetate but principally from water. Second, the nonexchangeable hydrogens of glucose (positions H-6,6 and H-1) are shown to be introduced to the odd CH 2 via the NAD(P)H pool used by both reductases involved in the elongation steps of the fatty acid chain. Third, it is proved that hydrogens removed at sites 9,10 and 12,13 during desaturation by ⌬ 9 -and ⌬ 12 -desaturases are pro-R, and that during these desaturation steps ␣-secondary kinetic isotope effects occur at the 9 and 12 positions and not at the 10 and 13 positions.Fatty acids are ubiquitous natural products involved in many key biological processes, including acting as components of membranes, as lipophilic modifiers, as fuel stores, and as precursors of intracellular messengers (1-3). Their biosynthesis, which is strictly conserved throughout higher organisms, can be separated into two parts as follows: the formation of the basic C16 unit, palmitoylCoA (C16:1), by the fatty-acid synthase complex (FAS) 2 and the subsequent modification of this chain by a range of elongases, desaturases, conjugases, hydroxylases, and epoxidases. Many of these steps involve stereoselective enzymatic reactions during which hydrogen atoms are inserted or eliminated.We have previously shown that this process leads to a distribution of 2 H in long chain fatty acids that is nonstatistical (4 -7). Thus, isotopic fractionation is seen to be introduced. Two features general to all organisms so far examined can be noted. First, the methylenic groups at even positions tend to be richer than those at odd positions. Second, one of the ethylenic groups present at the positions of desaturation is consistently impoverished relative to the methylenic sites, whereas the other is not.Thus, each hydrogen atom in the final product will have a ( 2 H/ 1 H) ratio that is representative of its initial origin, its passage through the FAS elongation steps, and its potential participation in post-elongation modification of the chain. To date, it has proved possible to exp...

The quantitative determination of isotopic (2H/1H)i ratios at natural abundance using the SNIF-NMR protocol is a well-known method for understanding the enzymatic biosynthesis of metabolites. However, this approach is not always successful for analyzing large solutes and, specifically, is inadequate for prochiral molecules such as complete essential unsaturated fatty acids. To overcome these analytical limitations, we use the natural abundance deuterium 2D NMR (NAD 2D NMR) spectroscopy on solutes embedded in polypeptide chiral liquid crystals. This approach, recently explored for measuring (2H/1H)i ratios of small analytes (Lesot, P.; Aroulanda, C.; Billault, I. Anal. Chem. 2004, 76, 2827-2835), is a powerful way to separate the 2H signals of all nonequivalent enantioisotopomers on the basis both of the 2H quadrupolar interactions and of the 2H chemical shift. Two significant advances over our previous work are presented here and allow the complete isotopic analysis of four mono- and polyunsaturated fatty acid methyl esters: methyl oleate (1), methyl linoleate (2), methyl linolenate (3), and methyl vernoleate (4). The first consists of using NMR spectrometers operating at higher magnetic field strength (14.1 T) and equipped with a selective cryoprobe optimized for deuterium nuclei. The second is the development of Q-COSY Fz 2D NMR experiments able to produce phased 2H 2D maps after a double Fourier transformation. This combination of modern hardware and efficient NMR sequences provides a unique tool to analyze the (2H/1H)i ratios of large prochiral molecules (C-18) dissolved in organic solutions of poly(gamma-benzyl-L-glutamate) and requires smaller amounts of solute than previous study on fatty acids. For each compound (1-4), all 2H quadrupolar doublets visible in the 2D spectra have been assigned on the basis of 2H chemical shifts, isotopic data obtained from isotropic quantitative NAD NMR, and by an interspectral comparison of the anisotropic NAD spectra of four fatty acids. The NMR results are discussed in terms of (2H/1H)i isotopic distribution and molecular orientation in the mesophase. For the first time, we show that the investigation of natural isotopic fractionation of complete fatty acids is possible without the need of chemical modifications, hence providing an alternative method to probe the mechanisms of enzymes implied in the biosynthetic pathway of unsaturated fatty acids.