Gamma-linolenic acid biosynthesis: cryptoregiochemistry of delta6 desaturation

The valuable nutraceutical gamma-linolenic acid (GLA, (6Z,9Z,12Z)-octadecatrienoic acid) is biosynthesized by a series of regio- and stereoselective dehydrogenation reactions that are catalyzed by a set of enzymes known as fatty acid desaturases. As part of ongoing research into the mechanism of these remarkable catalysts, we have examined the cryptoregiochemistry (site of initial oxidation) of Delta6 desaturation as it occurs in the protozoan Tetrahymena thermophila. Two complementary approaches that address this issue are described. In the first set of experiments, we measured the individual primary deuterium kinetic isotope effects associated with the C-H bond cleavages at C-6 and C-7. Competition experiments using appropriately deuterium-labeled 4-thiasubstrates revealed that a large KIE (kH/kD = 7.1 +/- 0.5) was observed for the C-H bond-breaking step at C-6, whereas the C-H bond cleavage at C-7 was insensitive to deuterium substitution (kH/kD = 1.04 +/- 0.05). These results point to C-6 as the site of initial oxidation in Delta6 desaturation since the first chemical step in this type of reaction is rupture of a strong, unactivated C-H bond, an energetically difficult process that typically exhibits a large KIE. This conclusion was supported by the results of our second approach, which involved locating the position of the putative diiron oxo oxidant with respect to substrate by monitoring the efficiency of oxo transfer to a series of thia fatty acid probes. Thus only a 6-thia-analogue is converted to significant amounts of the corresponding sulfoxide (9% yield). The absolute configuration of this product was determined to be S using (S)-MPAA as a chiral shift reagent. Taken together, these results point to the abstraction of the C-6 pro S hydrogen as the initial event in Delta6 desaturation as it occurs in T. thermophila.