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.

The intermolecular primary deuterium isotope effects on the individual C−H bond cleavage steps involved in linoleic acid biosynthesis were determined using a suitably transformed strain of Saccharomyces cerevisiae containing a functional oleate Δ12 desaturase from Arabidopsis thaliana. Mass spectral analysis of the methyl 7-thialinoleate fraction obtained from competition experiments involving methyl 7-thiastearate, methyl [12,12-2H2]-7-thiastearate and methyl [13,13-2H2]-7-thiastearate showed that cleavage of the C12−H bond is very sensitive to isotopic substitution (k H/k D = 7.3 ± 0.4) while a negligible isotope effect (k H/k D = 1.05 ± 0.04) was observed for the C13−H bond breaking step. This result strongly suggests that the site of initial oxidation for Δ12 desaturation is at C-12. The possible relationship between castor oleate 12-hydroxylase and microsomal Δ12 oleate desaturases is discussed in the context of a common mechanistic paradigm. Our methodology may be also be useful in deciphering the cryptoregiochemistry of other desaturase systems.

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The diiron center in stearoyl-acyl carrier protein (ACP) desaturase (DS) from castor plant Ricinus communis catalyzes the dioxygen- and NADPH-dependent introduction of a cis double bond between C9 and C10 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77 K produces an electron paramagnetic resonance (EPR)-detectable mixed-valence center (or [DS(ox)](mv)) that is trapped in the conformation of the diferric precursor and thus provides a sensitive EPR/electron nuclear double resonance (ENDOR) probe of the structure of the diamagnetic diiron(III) state. The cryoreduced DS shows two distinct EPR signals, suggesting the presence of two diiron(III) states: the mu-oxo (major)- and mu-hydroxo (minor)-bridged diiron centers. ENDOR studies show that in the dominant oxo-bridged diferric state each iron(III) coordinates a histidine and a water along with other ligands. Samples containing stoichiometric amounts of stearoyl-ACP show pronounced changes in the EPR and (1)H ENDOR spectra of cryoreduced DS. EPR spectra of the cryoreduced DS-substrate complex reveal two distinct substates of the parent. EPR and ENDOR studies show that both major conformers of the diferric cluster have a mu-oxo bridge. ENDOR shows that the major conformer has a histidine and a water bound to both Fe ions. In the minor conformer, one of the irons has lost the terminal water ligand. The structure of the trapped [DS(ox)](mv) state relaxes upon annealing to 170 K: the mu-oxo bridge in the major cryoreduced DS species protonates on annealing to 170 K; this does not occur for the major DS-substrate complex, even upon annealing to 230 K. The relaxed Fe(II)Fe(III) center in cryoreduced DS and DS-substrate show much less intense and resolved (14)N ENDOR spectra than those of the structurally similar cryoreduced diiron center in ribonucleotide reductase (RNR) protein R2. This difference may reflect some differences in His-Fe bonds. The alterations in the diferric site of DS induced by substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the diferric site. The mixed-valence states of DS are unstable at temperatures above 230 K.