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 Delta(9)- and Delta(12)-desaturases are pro-R, and that during these desaturation steps alpha-secondary kinetic isotope effects occur at the 9 and 12 positions and not at the 10 and 13 positions.
Highlights
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 [1,2,3]
Culture of F. lateritium and Isolation of Methyl Linoleate— Fatty acids were extracted as their methyl esters from cultures of the oleaginous filamentous fungus F. lateritium grown in media supplemented with different slightly enriched sources of 2H: water, glucose, or acetate (Table 1, cultures B–E)
Determination of (2H/1H) Ratios of Methyl Linoleate by Quantitative 2H NMR in Liquid and Chiral Oriented Media— The Q-COSY Fz map recorded in the chiral liquid crystal mesophase shows that numerous 2H sites that had coincident resonances in the isotropic 2H-{1H} NMR spectrum are clearly separated on the basis of a quadrupolar splitting difference
Summary
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 [1,2,3]. It has proved possible to explain part of the observed (2H/1H) ratios on the basis of the described mechanisms and measured isotope effects of the enzymes involved in these reactions In this way, it has been shown that the hydrogens at odd positions are essentially derived from NAD(P)H [8, 9], whereas those at even positions are derived in a very variable ratio from acetate and water (10 –12) (Fig. 1). The distribution pattern of 2H in a product can be accessed directly by quantitative 2H-{1H} NMR in isotropic media at natural abundance or at a level of enrichment in 2H too low to induce any significant isotope effect (typically 2–5-fold is used) The links between these values measured in the product and those for the various available origins of hydrogen can be quantified using a simple linear model, which describes the amount of hydrogen at each position derived from water and JOURNAL OF BIOLOGICAL CHEMISTRY 10783
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