The biosynthesis of dehydrodolichyl phosphates by rat liver microsomes

Using improved conditions with rat liver microsomes in the presence of 20% glycerol and 2% Triton X-100 at pH 8.5 it was shown that dehydrodolichyl diphosphate and dehydrodolichyl phosphate were synthesized from isopentenyl diphosphate and farnesyl diphosphate. Small amounts of geranylgeranyl diphosphate and geranylgeranyl phosphate were also formed. The carbon chain lengths of the dehydrodolichyl diphosphate and dehydrodolichyl phosphate were identical (C₈₀C₈₅). A kinetic study showed that dehydrodolichyl diphosphate formed from farnesyl diphosphate and isopentenyl diphosphate was subsequently hydrolyzed to dehydrodolichyl phosphate. As the concentration of isopentenyl diphosphate was increased from 1 to 50μ M, the chain-length distribution of dehydrodolichyl products shifted from C₇₅C₈₀ to C₈₀C₈₅. Addition of MgCl₂ into the assay mixture decreased product formation, but did not affect the chain-length distribution (C₈₀C₈₅). The shift of the chain-length distribution to the same as that observed in naturally occurring dolichol derivatives (C₉₀C₉₅) was observed when Triton X-100 was omitted from the assay mixture, although deletion of the detergent decreased the enzyme activity. These results, which provide insight into optimal conditions for enzymatic synthesis of the dolichol chain, are discussed in the context of the in vivo pathway for dolichol biosynthesis.