The biological syn-dehydrogenation (desaturation) of fatty acids 1 as exemplified by the ∆9 desaturase-mediated transformation of stearoyl CoA (1) to give oleyl CoA (2) represents one of the more virtuosic displays of enzymatic selectivity. Two classes of desaturases catalyze this intriguing transformation: soluble plant enzymes containing a carboxylate-bridged, nonheme diiron center 2 and less well-characterized, nonheme iron, membrane-bound catalysts as represented by the ∆9 desaturase found in Saccharomyces cereVisiae 3 and rat liver. 4 In light of the mounting evidence 5 that desaturases and hydroxylases are structurally related at the protein level, we have adopted the view that desaturations are initiated by a hydrogen abstraction step similar to that proposed for biohydroxylation. Some of the possible subsequent steps to the olefin are outlined in Scheme 1. 6 We have focussed our attention on the ∆9 desaturase of S. cereVisiae and have tentatively placed the putative iron-oxo oxidizing species near C-9 of the substrate since this enzyme system consistently oxygenates 9-thia fatty acid analogues more efficiently than the corresponding 10-thia analogues. 12 In this communication, we report the results of a study in which we further investigate the cryptoregiochemistry of yeast ∆9 desaturation by measuring the deuterium isotope effect for each individual C-H bond cleavage. 13 In order to expedite our isotope effect study, we decided to run direct competition experiments involving methyl 7-thiastearate-9,9-d 2 (3-9,9-d 2 ) vs methyl 7-thiastearate (3) and methyl 7-thiastearate-10,10-d 2 (3-10,10-d 2 ) vs methyl 7-thiastearate (3). Use of methyl stearate-d 2 /d 0 mixtures would have complicated the analysis of the methyl oleate-d 1 /d 0 product due to mass spectral interference by endogenous (d 0 ) oleate. The sulfur atom was placed at position 7 in order to facilitate the synthesis of the deuterated substrates. We have shown previously that methyl 7-thiastearate (3) is converted to the corresponding thiaoleate product (4). 14 3-9,9-d 2 and 3-10,10-d 2 were synthesized in 10% and 8% overall yield, respectively, using well-known procedures as shown in Scheme 2. 15 The two deuterated substrates consisted entirely of dideuterated species (within experimental error) as determined by MS; the 1 H and 13 C NMR spectra were consistent with the location of deuterium label. 3 was available from our previous study. A ca. 1:1 mixture of each deuterated substrate and d 0 material (25 mg) was administered as 5% w/v ethanolic solutions to growing cultures (150 mL) of S. cereVisiae ATCC 12341 as previously described. 12 Each incubation was carried The putative radical intermediate 7 could follow at least three different pathways: one-electron oxidation/H + elimination (pathway a), 8a,b disproportionation (pathway b), 9 or a hydroxyl rebound 10a (SH2) 10b /fast Fe 3+promoted dehydration sequence (pathway c). In addition, the possibility that organoiron intermediates 11 (not shown) are involved in these reactions cannot be...

The novel product profile obtained by incubating chiral fluorinated substrate analogues with castor stearoyl-ACP Delta(9) desaturase has been rationalized through a series of labeling studies. It was found that the introduction of the Z-double bond between C-9 and C-10 of the parent substrate occurs with pro-R enantioselectivity--a result that accounts for the observed stereochemistry of oxidation products derived from (9R)- and (9S)-9-fluorostearoyl-ACP. Oxidation of (9R)-9-fluorostearoyl-ACP occurs via at least two rapidly interchanging substrate conformations in the active site as detected by reaction pathway branching induced by deuteration at C-10 and C-11. Hydroxylation and desaturation of this substrate share the same site of initial oxidative attack.

No abstract