Abstract
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.
Highlights
To(S)-glycerylphosphate(GP)in two steps.Thefirst is catalyzed by geranylgeranylglyceryl phosphate (GGGP) synthase, an enzyme which attaches the geranylgeranyl moiety totheC[3] glyceryl hydroxyl inGP,andthe second by digeranylgeranylglycerylphosphate synthase,which alkylates the C(2) hydroxyl
Archaebacterial membranes contain a core lipid consisting of saturated Czoisopranyl glyceryl ethers [5, 10] instead of thefatty acid ester lipids foundinotherorganisms.This feature is unique to archaebacteria and places a substantial commitment on the isoprene pathwayfor synthesis of diterpenes
Tooursurprise, we discovered thatthe two activities copurified and subsequently were able to demonstrate that a single, bifunctional enzyme produces Farnesyl diphosphate (FPP) and GGPP in M. thermoautotrophicum
Summary
(Received for publication, November 30, 199a2n,d in revised form, January 25, 1993). Anjun Chenand 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. The abbreviations used are: GGPP, geranylgeranyl diphosphate; BHDA, bicyclo[2.2.1]hept-5-ene-2,3-dicarboxyliacnhydride; BME, p - mercaptoethanol; CAPS, 3-(cyclohexylamino)propanesulfonicacid DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GC, gas chromatography; GCMS, gas chromatography mass spectrometry; GGGP, geranylgeranylglyceryl phosphate; GP, glyceryl phosphate; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; MOPS, 3(N-morpho1ino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PIPES, piperazine-N,N”bis(2-ethanesulfonicacid).
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