Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

Abstract

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...

Analysis of NAD 2D‐NMR Spectra of Saturated Fatty Acids in Polypeptide Aligning Media by Experimental and Modeling Approaches

“…For C14 to C18 fatty acid methyl esters (FAMEs), the rate of discrimination is around 85 % of all accessible sites (14 to 18) in the achiral liquid crystals (PBG/Pyr), whereas this rate was only around 30 % in isotropic solvents. In the chiral mesophase (PBLG/Pyr) around 70 % of all accessible sites (26 to 34) could be discriminated; this proves again the intrinsic efficiency of NAD 2D-NMR in chiral media to resolve 2 H NMR resonances never observed so far. As a consequence, we have now in hands a tool able to establish almost entirely the natural isotopic profile of SAFAs and determine the eie (enantio-istopomeric excess) along the chain, as we already achieved for methyl linoleate.…”

“…. [2] Moreover, our analysis of SAFAs has greatly benefited from the knowledge gained from the previous studies on PUFAs. [1,6] Figure 4 schematically depicts our analytical approach.…”

“…Computational methods: The S CÀD order parameter in Equation (1) is defined in Equation (2), in which the angular bracket denotes the statistical average and q CÀD is the angle between the C À D bond and the liquid crystal director, that is the average orientation of the polypeptide axis, which is parallel to the magnetic field axis (B o ) of the NMR spectrometer.…”

“…[1][2][3][4][5][6][7] The method allows to reach important information, related to the stereochemistry of analytes, unrevealed by the classical SNIF NMR method proposed by Martin and co-workers. [8] The alternative approach was successfully applied for analyzing monoor polyunsaturated (diene or triene) fatty acids (PUFAs).…”

Hydrogen dynamics in soil organic matter as determined by <sup>13</sup>C and <sup>2</sup>H labeling experiments

“…It is in accordance with the work of Baillif and colleagues that have grown fungi with labeled glucose, water and acetate to trace the incorporation of 2 H during fatty acid biosynthesis. They have demonstrated that water is the main donor of hydrogen atoms in the non-exchangeable pool within the biosynthesis cycle (Baillif et al, 2009). Ruppenthal and colleagues have shown as well that precipitation contributes up to 80 % of the isotopic composition of non-exchangeable hydrogen (Ruppenthal et al, 2010).…”