gamma-linolenic acid biosynthesis: Cryptoregiochemistry of Delta(6) desaturation

The valuable nutraceutical gamma -linolenic acid (GLA, (6Z,9Z,12Z)-octadeca trienoic acid) is biosynthesized by a series of regio- and stereoselective dehydrogenation reactions that are catalyzed by a set of enzymes known as f atty acid desaturases. As part of ongoing research into the mechanism of th ese remarkable catalysts, we have examined the cryptoregiochemistry (site o f initial oxidation) of Delta (6) desaturation as it occurs in the protozoa n Tetrahymena thermophila. Two complementary approaches that address this i ssue are described. In the first set of experiments, we measured the indivi dual primary deuterium kinetic isotope effects associated with the C-H bond cleavages at C-6 and C-7. Competition experiments using appropriately deut erium-labeled 4-thiasubstrates revealed that a large KIE (k(H)/k(D) = 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 (k(H)/k(D) = 1.04 +/- 0.05). These results point to C-6 as the site of initial oxidati on in Delta (6) desaturation since the first chemical step in this type of reaction is rupture of a strong, unactivated C-H bond, an energetically dif ficult process that typically exhibits a large KIE. This conclusion was sup ported by the results of our second approach, which involved locating the p osition of the putative diiron oxo oxidant with respect to substrate by mon itoring the efficiency of oxo transfer to a series of thia fatty acid probe s. Thus only a 6-thia-analogue is converted to significant amounts of the c orresponding sulfoxide (9% yield). The absolute configuration of this produ ct was determined to be S using(S)-MPAA as a chiral shift reagent. Taken to gether, these results point tb the abstraction of the C-6 pro S hydrogen as the initial event in Delta (6) desaturation as it occurs in T. thermophila .