hydrogen at C2 of IPP is removed in the IPP f DMAPP
isomerization.4 We now report that FPP synthase from E.
coli, like the eukaryotic enzymes, also catalyzes the con-
densation of IPP with GPP by selectively removing the pro-R
hydrogen at C2 of IPP.
incubation was continued for 8 h. At the end of the
incubation, the voluminous white precipitate that had formed
was redissolved by gradual addition of 3 equiv of EDTA
with vigorous agitation. FPP from the reaction was purified
by preparative reverse phase HPLC on a C18 Shodex
Asahipack (Phenomenex) column11 and analyzed by 1H NMR
spectroscopy. An identical experiment was run in parallel
with chicken liver FPP synthase as control.
Figure 1 shows the low-field region of the spectrum of
purified FPP synthesized by the E. coli FPP synthase. Signals
at δ 4.52, 5.24, and 5.52 correspond to protons at C1, C6/
C10, and C2, respectively. The ratios of areas under the peaks
at δ 4.52 and 5.52 (4.52/5.52 ) 2.2) and peaks at δ 5.24
and 5.52 (5.24/5.52 ) 2.1) clearly show that little if any of
the deuterium initially present in the pro-R position at C2
of IPP was transferred to the product FPP. This result, which
is identical to that obtained with the control enzyme,
demonstrates that E. coli FPP synthase selectively removes
the pro-R hydrogen at C2 of IPP during the condensation
reaction.
Recombinant E. coli FPP synthase was obtained following
standard molecular biology techniques. The ispA gene which
encodes for FPP synthase was amplified from E. coli
genomic DNA by “sticky-end” PCR5 and ligated into a
pET11a expression vector6 utilizing NdeI and BamHI restric-
tion sites. The resulting plasmid was purified and transformed
into BL21(DE3) competent cells. Expression studies in small
scale cultures showed no difference in the total amount of
overexpressed protein and enzyme activity when the cells
were grown up in LB, superbroth, or minimal (M9) media.7
Preparative-scale cultures in LB were grown at 37 °C, and
plasmid-directed protein synthesis was induced by addition
of IPTG. Following an additional incubation period, cells
were harvested and disrupted. Overexpressed E. coli FPP
synthase was purified from cell extracts in two steps as
described previously for the avian enzyme.8
The experiment to determine the stereospecificity of the
condensation of IPP and GPP by E. coli FPP synthase is
outlined in Scheme 2. GPP (12 mg, 32.2 µmol) and
Scheme 2
Figure 1. 500 MHz 1H NMR spectrum of enzymatically produced
FPP, low-field region. Signal at δ 5.52 (vinylic proton at C2)
demonstrates deuterium was not incorporated into FPP. Broad signal
centered at δ 4.9 is the residual absorption of the solvent after
presaturation.
Isoprenoid biosynthesis is known to occur in E. coli by a
recently discovered methylerythrose phosphate (MEP)-de-
pendent pathway.12 The carbon atoms in the isoprene unit
come from glyceraldehyde and pyruvate. The two three-
carbon precursors are condensed with loss of CO2 to give
1-deoxy-D-xylulose phosphate 113,14 (Scheme 3). The phos-
phate 1 is then the substrate for a reductoisomerase15 that
catalyzes a skeletal rearrangement and reduction to produce
recombinant E. coli FPP synthase (0.57 unit)9 were preincu-
bated at 37 °C for 2 min in a 20 mM BHDA buffer, pH 7.0,
containing 25 mM MgCl2, 50 mM KCl, and 0.5 mM DTT.
A 5 mg (16.6 mmol) sample of (R)-[2-2H]isopentenyl
diphosphate10 was then added in three portions over the
course of 45 min (final reaction volume was 2.0 mL), and
(4) Leyes, A. E.; Baker, J. A.; Hahn, F. M.; Poulter, C. D. Chem.
Commun. 1999, 717.
(5) Zeng, G. Biotechniques 1998, 25, 206.
(11) Elution conditions: 5 mL min-1, isocratic 20% MeCN-80% 25
mM NH4HCO3 for 5 min and then linear gradient to 100% MeCN in 25
min.
(12) The MEP nomenclature is parallel to the MVA (mevalonate)
pathway.
(13) Rohmer, M.; Seeman, M.; Horbach, S.; Bringer-Meyer, S.; Sahm,
H. J. Am. Chem. Soc. 1996, 118, 2564.
(14) Sprenger, G.; Scho¨rken, U.; Wiegert, T.; de Graaf, A. A.; Taylor,
S. V.; Begley, T. P.; Bringer-Meyer, S.; Sahm, H. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 12857.
(6) Studier, F. W.; Rosenberg. A. H.; Dunn, J. J.; Dubendorff, J. W.
Methods Enzymol. 1990, 185, 60. Plasmid commercially available from
Novagen Inc., 601 Science Dr., Madison, WI 53711.
(7) For a description of culture media, see: Maniatis, T.; Fritsch, E. F.;
Sambrook, J. Molecular Cloning: A Laboratory Manual; Cold Spring
Harbor Laboratory Press: Cold Spring Harbor, NY, 1982.
(8) Tarshis, L. C.; Yan, M.; Poulter, C. D.; Sacchettini, J. C. Biochemistry
1994, 33, 10871.
(9) One unit is the amount of protein (mg) that can produce 1 µmol of
product in 1 min.
(10) Leyes, A. E.; Poulter, C. D. Org. Lett. 1999, 1, 1067-1070.
(15) Takahashi, S.; Kuzuyama, T.; Watanabe, H.; Seto, H. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9879.
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Org. Lett., Vol. 1, No. 7, 1999