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.
Chen
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Department of Chemistry, University of Utah, Salt Lake City 84112.
Poulter
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D019883:Alkyl and Aryl Transferases; D002413:Cations, Divalent / Q000494:pharmacology; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D051231:Farnesyltranstransferase; D006882:Hydroxyapatites; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology*; D008970:Molecular Weight; D013816:Thermodynamics; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism*
D002413:Cations, Divalent; D006882:Hydroxyapatites; D017886:Durapatite; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase