Abstract
Comparison of the farnesyl diphosphate (FPP) synthase amino acid sequences from four species with amino acid sequences from the related enzymes hexaprenyl diphosphate synthase and geranylgeranyl diphosphate synthase show the presence of two aspartate rich highly conserved domains. The aspartate motif ((I, L, or V)XDDXXD) of the second of those domains has homology with at least 9 prenyl transfer enzymes that utilize an allylic prenyl diphosphate as one substrate. In order to investigate the role of this second aspartate-rich domain in rat FPP synthase, we mutated the first or third aspartate to glutamate, expressed the wild-type and mutant enzymes in Escherichia coli, and purified them to apparent homogeneity using a single chromatographic step. Approximately 12 mg of homogeneous protein was isolated from 120 mg of crude bacterial extract. The kinetic parameters of the purified wild-type recombinant FPP synthase containing the DDYLD motif were as follows: Vmax = 0.84 mumol/min/mg; GPP Km = 1.0 microM; isopentenyl diphosphate (IPP) Km = 2.7 microM. Substitution of glutamate for the first aspartate (EDYLD) decreased the Vmax by over 90-fold. The Km for IPP increased, whereas the Km for GPP remained the same in this D243E mutant. Substitution of glutamate for the third aspartate (DDYLE) did not result in altered enzyme kinetics in the D247E mutant. These results suggest that the first aspartate in the second domain is involved in the catalysis by FPP synthase.
Highlights
8) and phytoene synthase(crtB) (9, lo), are bifunctional enzymes
We demonstrate that thekinetic parameters of the purified wildtype rat recombinant Farnesyl diphosphate (FPP) synthase aresimilar to the purified moiety of heme a [1]
In order to determine whether the conserved aspartates played a significant role in binding either the allylic or the homoallylic substrate or in the catalytic activity of the rat FPP synthase the normal and mutantenzymes, in which the glutamate had replaced an aspartate, were overexpressed in E. coli, purified, and assayed for enzymeactivity
Summary
The apparent molecular mass of kDa determined here for rat liver F P P synthase is consistent with the predicted molecular mass calculated from the synthase cDNA nucleotide sequence [17,18,19] Both mutant and wild-type FPP synthase were recognizedon immunoblots by antibodies raised against an FPPsynthase fusion protein [23] (data not shown). Wild-type and mutant FPP synthases were purified from crude cell-free extracts by ammonium sulfate precipitation followed byion exchangechromatographyon DEAE-cellulose. This procedure yielded proteins that gave a single band on SDS-PAGE and were judged to be greater than 95% pure (Fig. 3, lane 4 for wild-type, lane 6 for D243E mutant, and lane 8 for D247E mutant). Wild-type rat liver FPP synthase was overexpressed and resulted in at least a 360-fold higher specific activity than the endogenousE. coli FPP synthase activity, and more than 30% of the E. coli soluble protein was rat liver FPP synthase, as (A)
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