Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris

Abstract

The hypothesis that the Δ9 desaturase of Chlorella vulgaris might operate by a synchronous mechanism has been tested using a kinetic isotope effect (KIE) approach. Thus the intermolecular primary deuterium KIE on the individual C-H bond cleavage steps involved in Δ9 desaturation have been determined by incubating growing cultures of C. vulgaris (strain 211/8K) with mixtures of the appropriate regiospecifically deuterated fatty acid analogues. Our analysis shows that the introduction of a double bond between C-9 and C-10 occurs in two discrete steps as the cleavage of the C9-H bond is very sensitive to isotopic substitution (kH/kD = 6.6 ± 0.3) whereas a negligible isotope effect (kH/kD = 1.05 ± 0.05) was observed for the C10-H bond-breaking step. Similar results were obtained for linoleic acid biosynthesis (Δ12 desaturation). These data clearly rule out a synchronous mechanism for these reactions.