PURIFICATION AND CHARACTERIZATION OF GERANYLGERANYL DIPHOSPHATE SYNTHASE FROM METHANOBACTERIUM-THERMOFORMICICUM SF-4

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) = 30.8 muM) and either dimethylallyl diphosphate (K(m) = 16.8 muM), geranyl diphosphate (K(m) = 12.6 muM), or farnesyl diphosphate (K(m) = 14.7 muM) as allylic partners. During a sequential elongation, C5-->C-10-->C-15-->C20, intermediates were accumulated with various ratios to the final product GGPP. In the presence of 0.8 M KCl, GGPP synthease activity was greatly enhanced, stabilized to heat treatment at 65-degrees-C for 30 min, 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 synthase alone is important in the biosynthetic pathways to squalene and membrane polar lipids at a chain elongation stage in this strain.