Akemi Kinoshita

Novel pathways of oleic and cis-vaccenic acid biosynthesis by an enzymatic double-bond shifting reaction in higher plants

The novel pathways of oleic acid formation from cis‐vaccenic (cis‐11‐octadecenoic) acid and of cis‐vaccenic acid formation from oleic acid by enzymatic positional isomerization have been proposed in higher plants, based on stable‐isotope experiments using[2,2‐2H2]cis‐vaccenate or [2,2‐2H2]oleate as an immediate precursor. A pulp homogenate and also pulp slices prepared from developing kaki (Diospyros kaki) fruit could catalyze these hitherto unknown isomerizations. This suggests the presence of a new type of isomerase responsible for the double‐bond shifting reaction without cis‐trans isomerization in the middle of fatty acid carbon chains.

Application of a GC-MS method using deuterated fatty acids for tracingcis-vaccenic acid biosynthesis in kaki pulp

A gas chromatographic-mass spectrometric method using [2,2-2H2]fatty acids has been developed to trace the biosynthesis ofcis-vaccenic (cis-11-octadecenoic) acid in higher plants. The deuterated fatty acids and other unlabeled fatty acids in the biosynthetic reaction mixture were converted into bis(methylthio) derivatives and analyzed by mass chromatography. The principle of this method was based on the shift of key fragment ions (containing two deuterium atoms) due to the cleavage between the methylthio-substituted carbons. The labeled compounds were detected by the m/z values which shifted 2 mass units from those of the corresponding unlabeled compounds and estimated by a calibration curve based on the peak areas of the key fragment ions. For metabolic experiments, a homogenate fraction was prepared from the pulp part of maturing kaki (Diospyros kaki) fruit and incubated with ammonium [2,2-2H2]palmitoleate (cis-9-hexadecenoate) or [2,2-2H2]palmitoleoyl-CoA. The incubation resulted in the formation of detectable amounts of isotopically-labeledcis-vaccenic acid containing two deuterium atoms at the carbon chain between the double bond and the carboxyl group. This experimental evidence proved thatcis-vaccenic acid was formed from palmitoleic acid by chain elongation.