Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis

Abstract

The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 μ m, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 m m. The product of the reaction has been characterized as an α-unsaturated polyprenyl pyrophosphate, containing 12–15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of α- cis- and α- trans-pentadecaprenyl phosphates.