Diphosphate Synthase Research Articles

Purification and characterization of farnesyl diphosphate/geranylgeranyl diphosphate synthase. A thermostable bifunctional enzyme from Methanobacterium thermoautotrophicum

Farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase, a bifunctional enzyme that synthesizes C15 and C20 isoprenoid diphosphates from isopentenyl diphosphate and dimethylallyl diphosphate, was purified to homogeneity from the archaebacterium Methanobacterium thermoautotrophicum. The only activities detected from synthesis of FPP and GGPP copurified through (NH4)2SO4 precipitation and four chromatographic steps. The pure enzyme was a 79-kDa homodimer that catalyzed the sequential addition of isopentenyl diphosphate to dimethylallyl diphosphate, geranyl diphosphate, and FPP by a non-processive mechanism which allowed substantial amounts of FPP to accumulate during turnover, creating a pool for further elongation to GGPP or for synthesis of squalene. The bifunctional enzyme required Mg2+ or Mn2+ and was optimally active at 65 degrees C. Catalysis of chain elongation in M. thermoautotrophicum differs from related reactions in eubacteria and eukaryotes, where distinct FPP synthases and GGPP synthases are found.

Distribution of prenyltransferases in rat tissues. Evidence for a cytosolic all-trans-geranylgeranyl diphosphate synthase.

The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synthase activities, one cytosolic and one membrane-associated, in a number of rat tissues. Both enzymes utilize farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) as substrates, but they give rise to different products. The membrane-associated activity produces trans,trans,cis-(E,E,Z)-GGPP, involved in the biosynthesis of long-chain polyprenols. The cytosolic activity produces only the all-trans-(E,E,E) isomer of GGPP, which is utilized as substrate in cytosolic protein prenylation reactions. All-trans-GGPP synthase activity was recovered in the cytosolic fraction from all tissues investigated, but the specific activities varied. The highest specific activities were found in brain, spleen, and testis, followed by kidney and liver. The enzyme activity in rat brain cytosol was further characterized and found to exhibit a narrow pH optimum around 5.0-6.0 and to be highly stimulated by Zn2+. Maximal stimulation was attained with 1 mM Zn2+, whereas Mg2+ had no effect on the enzyme activity. The all-trans-GGPP synthase activity exhibited high affinities for its substrates, i.e. the apparent Km values for FPP and IPP were found to be 0.6 and 3.5 microM, respectively. When rats were fed mevinolin (lovastatin), FPP and all-trans-GGPP synthase activities were affected differently in certain tissues. Mevinolin treatment resulted in an increase in FPP but a decrease in all-trans-GGPP synthase activity in rat liver and kidney. In spleen mevinolin treatment caused a greater than 70% decrease in all-trans-GGPP synthase activity, while FPP synthase was almost unaffected. The presence of two different GGPP synthase activities in the cell, together with the fact that FPP and all-trans-GGPP synthesis in the cytosol are regulated independently, may be of significance in the regulation of isoprenoid biosynthesis, as well as of protein isoprenylation.