Molecular cloning and characterization of a membrane-intrinsic (S)‎-2,3-di-O-digeranylgeranylglyceryl phosphate synthase involved in the biosynthesis of archaeal ether-linked membrane lipids

الملخص EN

The second step in the biosynthesis of the core membrane diether lipids in archaea is synthsis of digerany1gerany1glycery1 phosphate from gerany1gerany1glycery1 phosphate and gerany1geranyl pyrophosphate.

The reaction is catalyzed by (S)-2,3-di-O-gerany1gerany1g1ycer1y phoshate synthase (DGGGP synthase).

The gene encoding the DGGGP synthase was cloned from Methanocaldococcus jannaschii (MJ 0279) and expressed in the cells of Escherchia coli C41 (DE3).

The membrane protein was then solubilized by 2% n-Octyl-β-D-g1ucopyranoside and purified to homogeneity by a combination of heat treatment, DEAE-Sepharose, Resource Q and Hydroxyapatite column chromatography.

The native polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis of purified DGGGPS gave a single band at 30 kDa.

The optimum temperature and pH of the purified enzyme was 70°C and pH 6.0, respectively.

The enzyme requires Mg2+ for optimal activity, while EDTA inhibits the activity.

Other characteristics, including substrate specificity, salt effects and detergents effects were determined.