Partial purification and characterization of the short-chain prenyltransferases, geranyl diphosphate synthase and farnesyl diphosphate synthase, from Abies grandis (grand fir)

In the conifer Abies grandis (grand fir), a secreted oleoresin rich in mono-, sesqui-, and diterpenes serves as a constitutive and induced defense against insects and pathogenic fungi. Geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) synthase, two enzymes which form the principal precursors of the oleoresin mono- and sesquiterpenes, were isolated from the stems of 2-year-old grand fir saplings. These enzymes were partially purified by sequential chromatography on DEAE-Sepharose, Mono-Q, and phenyl-Sepharose to remove competing phosphohydrolase and isopentenyl diphosphate (IPP) isomerase activities. GPP and FPP synthase formed GPP and E,E-FPP, respectively, as the sole products of the enzymatic condensation of IPP and dimethylallyl diphosphate (DMAPP). The properties of both enzymes are broadly similar to those of other prenyltransferases. The apparent native molecular masses are 54 +/- 3 kDa for GPP synthase and 110 +/- 6 kDa for FPP synthase, as determined by gel permeation chromatography. Michaelis-Menten constants for GPP synthase are 14.3 and 16.7 muM for IPP and DMAPP, respectively. For IPP synthase, K(m) values of 15.3 and 9.0 muM were determined for IPP and DMAPP, respectively, and the K(m) value for GPP was 1.8 muM. Both enzymes require a divalent metal ion for catalytic activity. GPP synthase prefers Mg(2+) as cofactor, whereas in the case of FPP synthase, Mg(2+) can be substituted almost equivalently by Mn(2+), Zn(2+), or Co(2+). The products of both prenyltransferase reactions, including inorganic diphosphate, GPP, and FPP, are inhibitory. No significant induction of either enzyme activity was observed in stem tissue over a 20-day time course after mechanical wounding, (C) 2001 Academic Press.