Deciphering the Cryptoregiochemistry of Oleate Δ¹² Desaturase:  A Kinetic Isotope Effect Study

Abstract

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.

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

“…Such an ␣-secondary KIE has not previously been recorded for these desaturases. However, it is fully consistent with previous studies showing a primary KIE at site 9 but not at site 10 during the action of ⌬ 9 -desaturase (42) and at site 12 but not at site 13 during the action of ⌬ 12 -desaturase (43). Interestingly, the deuterium profile measured by 2 H NMR in chiral oriented media of methyl linoleate isolated from a plant (sunflower) shows similar ( 2 H/ 1 H) 9 and ( 2 H/ 1 H) 13 ratios, whereas the ( 2 H/ 1 H) 10 ratio was higher than the ( 2 H/ 1 H) 12 ratio (24).…”

Natural Deuterium Distribution in Fatty Acids Isolated from Peanut Seed Oil: A Site-Specific Study by Quantitative 2H NMR Spectroscopy

“…A strong KIE has also been reported at C12 during D 12 desaturation. [31,32] In this study, in contrast, no secondary component was found for this position. Rather, a secondary isotope effect of similar size to that observed for the C9 position during D 9 desaturation was measured at C13, at which negligible isotope effect was reported.…”

“…Rather, a secondary isotope effect of similar size to that observed for the C9 position during D 9 desaturation was measured at C13, at which negligible isotope effect was reported. [31,32] This discrepancy indicates that, by observing natural abundance isotopic parameters, it may be possible to obtain isotopic fractionation data not visible by classical enrichment techniques.…”

Desaturation reactions catalyzed by soluble methane monooxygenase

“…A similar conclusion was reached from studies of D9D in which it was shown that mutagenesis of only a few key residues resulted in a switch from desaturation to hydroxylation, just the opposite shift in the primary reaction chemistry as observed here for MMO [58]. Similarly, isotope eect studies of the reaction of Arabidopsis thaliana D 12 -desaturase supported the hypothesis that hydrogen atom abstraction yields a substrate (2) oxygen radical rebound and epoxide formation; (3) hydrogen abstraction; (4) hydroxylation; (5) radical resonance; (6) blocked hydroxylation step; (7) electron abstraction and elimination radical intermediate which can proceed to the desaturated product by either simple disproportionation or a two-step oxidation/deprotonation, as we propose here [59]. It is important to note that the desaturase enzymes yield dehydrogenated products without special stabilization of the position that is attacked in the substrate as required in the case of MMO.…”