Purification and Characterization of Geranylgeranyl Diphosphate Synthase from Methanobacterium thermoformicicum SF-4

Abstract

Geranylgeranyl diphosphate (GGPP) synthase [EC 2.5.1.29] was purified to homogeneity from Methanobacterium thermoformicicum SF-4. The enzyme was a dimeric protein consisting of two identical subunits (M_r=39, 000) and catalyzed prenyl transfer reactions using isopentenyl diphosphate (K_m=30.8μM)and either dimethylallyl diphosphate (K_m=16.8μM), geranyl diphosphate (K_m=12.6μM), or farnesyl diphosphate (K_m=14.7μM) as allylic partners. During a sequential elongation, C₅→C₁₀→C₁₅→C₂₀, intermediates were accumulated with various ratios to the final product GGPP. In the presence of 0.8M KCl, GGPP synthease activity was greatly enhanced, stabilized to heat treatment at 65°C for 30min, and protected from inhibition by p-chloromercuribenzoic acid. No other prenyltransferase synthesizing C₂₀ or shorter prenyl diphosphate was observed in M. thermoformicicum SF-4. These suggest that GGPP synthease alone is important in the biosynthetic pathways to squalene and membrane polar lipids at a chain elongation stage in this strain.