Partial purification and characterization of the short-chain prenyltransferases, geranyl diphosphate synthase and farnesyl diphosphate synthase, fromAbies grandis (grand fir)
D. Tholl et al., Partial purification and characterization of the short-chain prenyltransferases, geranyl diphosphate synthase and farnesyl diphosphate synthase, fromAbies grandis (grand fir), ARCH BIOCH, 386(2), 2001, pp. 233-242
In the conifer Abies grandis (grand fir), a secreted oleoresin rich in mono
-, sesqui-, and diterpenes serves as a constitutive and induced defense aga
inst insects and pathogenic fungi. Geranyl diphosphate (GPP) and farnesyl d
iphosphate (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 seq
uential chromatography on DEAE-Sepharose, Mono-Q, and phenyl-Sepharose to r
emove competing phosphohydrolase and isopentenyl diphosphate (IPP) isomeras
e activities. GPP and FPP synthase formed GPP and E,E-FPP, respectively, as
the sole products of the enzymatic condensation of IPP and dimethylallyl d
iphosphate (DMAPP). The properties of both enzymes are broadly similar to t
hose of other prenyltransferases. The apparent native molecular masses are
54 +/- 3 kDa for GPP synthase and 110 +/- 6 kDa for FPP synthase, as determ
ined by gel permeation chromatography. Michaelis-Menten constants for GPP s
ynthase are 14.3 and 16.7 muM for IPP and DMAPP, respectively. For IPP synt
hase, K-m values of 15.3 and 9.0 muM were determined for IPP and DMAPP, res
pectively, and the K-m value for GPP was 1.8 muM. Both enzymes require a di
valent metal ion for catalytic activity. GPP synthase prefers Mg2+ as cofac
tor, whereas in the case of FPP synthase, Mg2+ can be substituted almost eq
uivalently by Mn2+, Zn2+, or Co2+. The products of both prenyltransferase r
eactions, including inorganic diphosphate, GPP, and FPP, are inhibitory. No
significant induction of either enzyme activity was observed in stem tissu
e over a 20-day time course after mechanical wounding, (C) 2001 Academic Pr
ess.