Báo cáo khoa học: Biosynthesis of isoprenoids A bifunctional IspDF enzyme fromCampylobacter jejuni pot

Biosynthesis of isoprenoids
A bifunctional IspDF enzyme from
Campylobacter jejuni
Mads Gabrielsen
1
, Felix Rohdich
2
, Wolfgang Eisenreich
2
, Tobias Gra¨ wert
2
, Stefan Hecht
2
, Adelbert Bacher
2
and William N. Hunter
1
1
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, UK;
2
Lehrstuhl fu
¨
r Organische Chemie und Biochemie, Technische Universita
¨
tMu
¨
nchen, Garching, Germany
In the nonmevalonate pathway of i soprenoid biosynthesis,
the conversion of 2 C-methyl-
D
-erythritol 4-phosphate into

its cyclic diphosphate proceeds via nucleotidyl intermediates
and is catalyzed by the products of the ispD, ispE and ispF
genes. An open reading frame of Campylobacter jejuni
with similarity to the ispD and ispF genes of Escherichia coli
was cloned into an expression vector directing the formation
of a 42 k Da protein in a recombinant E. coli strain. The
purified protein w as shown t o c atalyze the transformation
of 2C-met hyl-
D
-erythritol 4-phosphate into 4-diphospho-
cytidyl-2C-met hyl-
D
-erythritol and the conversion of
4-diphosphocytidyl-2C-methyl-
D
-erythritol 2-phosphate
into 2C-methyl-
D
-erythritol 2,4-cyclodiphosphate at cata-
lytic rates of 19 lmol Æmg
)1
Æmin
)1
and 7 lmolÆmg
)1
Æmin
)1
,
respectively. Both enz yme-catalyzed reactions require diva-
lent metal i ons. The C. jejuni enzyme does not catalyze the

formation of 2C-methyl-
D
-erythritol 3,4-cyclophosphate
from 4-diphosphocytidyl-2C-methyl-
D
-erythritol, a side
reaction catalyzed in vitro by the IspF proteins of E. coli
and Plasmodium falciparum. Comparative genomic analysis
show that all sequenced a-ande-proteobacteria have fused
ispDF genes. These bifunctional proteins a re potential drug
targets in several human pathogens (e.g. Helicobacter pylori,
C. jejuni and Treponema pallidum).
Keywords: b ifunctional e nzyme; biosynthetic pathway;
isoprenoid; NMR; nonmevalonate.
Isoprenoids are one of the largest groups of n atural
products comprising numerous compounds with important
roles in physiological and pathological processes [1]. The
early work with yeast and animal cells established t he
biosynthesis of the two universal isoprenoid building blocks,
isopentenyl diphosphate (9, F ig. 1 ) a nd dim ethylallyl
diphosphate (10, F ig. 1), f rom acetyl-CoA via mevalonate
[2–5]. This pioneering work culminated in the development
of important drugs, most notably inhibitors of 3-hydroxy-3-
methylglutaryl-CoA reductase inhibitors (statins), which
are now widely used for the prevention and therapy of
cardiovascular disease [ 6,7].
About a decade ago, a second pathway for the f ormation
of 9 and 10 from pyruvate (1)and
D
-glyceraldehyde

3-phosphate ( 2) v ia 1- deoxy-
D
-xylulose 5 -phosphate (3)
was found to be operative in certain bacteria as well as in the
plastids of plants and protozoa [8–12]. The first committed
intermediate of this pathway, 2C-methyl-
D
-erythritol
4-phosphate (4), is formed from 3 by a skeletal r earrange-
ment followed by reduction; both reaction steps are
catalyzed by the IspC protein (Fig. 1) [13]. The polyol 4 is
converted into t he cyclic diphosphate 7 via 4-diphospho-
cytidyl-2C-met hyl-
D
-erythritol (5) and its 2 -phosphate 6 by
the consecutive action of three enzymes specified by the
ispD, E and F genes [14–16]. The conversion of 7 into 9 and
10 via t he recently identified intermediate 1-hydroxy-2-
methyl-2-(E)-butenyl 4-diphosphate (8) requires IspG and
IspH proteins [ 17–24].
The nonmevalonate pathway for the biosynthesis of
terpenoids is essential in a wide variety of pathogenic bacteria
including Mycobacterium tuberculosis and Mycobacterium
leprae as well as in the v arious Plasmodium species causing
malaria. Recent studies have already shown that malaria can
be treated by the antibiotic fosmidomycin, an inhibitor of the
IspC protein [25,26]. Hence, it appears safe to c onsider the
enzymes o f the nonmevalonate pathway as estab lished
therapeutic targets. T he detailed c haracterization of a ll
enzymes of this pathway is required in order to provide a firm

basis for the screening and development of novel drugs. Here
we describe the catalytic properties of the bifunctional IspDF
protein f rom t he human pathogen, Campylobacter jejuni.
Experimental procedures
Materials
[1,3,4-
13
C
3
]4 was prepared as described earlier [27]. The
preparation of [ 1,3,4-
13
C
3
]6 will be described elsewhere
(A. Bacher, F. Rohdich, W. Eisenreich, E. Ostrojenkova,
J. Kaiser & T. Gra
¨
wert, unpublished data). Sodium
[1,2-
13
C
2
]acetate was purchased from Isotec (Miamisburg,
OH, USA). CTP, ATP, phosphoenolpyruvate and pyruvate
kinase were obtained from S igma. Oligonucleotides were
custom synthesized by Sigma Genosys. Restriction enzymes,
Pfu polymerase and DNA ligase w ere purchased from
Promega. DNaseI was obtained f rom Roche. Protease
Correspondence to W . N. Hunter, Division of B iological Chemistry

and Molecular Microbiology, Sc hool of Life Sciences, Uni versity of
Dundee, Dundee, DD1 5EH, UK. Fax: + 44 1382 345764,
Tel.: + 44 1382 345745, E-mail:
(Received 17 March 2004, revised 25 M ay 2004,
accepted 28 May 2004)
Eur. J. Biochem. 271, 3028–3035 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04234.x
inhibitor cocktail was from Roche. Thr ombin was obtained
from Amersham Pharmacia Biotech. Recombinant IspE
protein from Escherichia coli was prepared as described
earlier [ 15].
Microorganisms and plasmids
Bacterial strains and plasmids used in this study are s hown
in Table 1.
Cloning and expression of the
ispDF
gene
from
C. jejuni
The putative ispDF open r eading frame was amplified fr om
bp position 49 226–50 340 (GenBank accession number
AL139079) by PCR using the oligonucleotides IspDF
Forward and IspDF Reverse (Table 2) as primers and
C. jejuni DNA as template. The amplicon, comprising
engineered restriction sites for NdeIandBamHI, was cloned
into the pCR-Blunt II TOPO vector [28] (Invitrogen).
Positive clones were identified by d igestion with Nde Iand
BamHI affording a 1.1 kb fragment that was cloned into the
pET15b expression vector (Novagen). The integrity of the
cloned gene was confirmed by sequencing. The resulting
plasmid pET15b-ispDF was heat-shock transformed into

the E. coli strain BL21 (DE3) [29], giving the recombinant
strain BL21-pET15b-ispDF.
Enzyme purification
The recombinant E. coli strain BL21-pET15b-ispDF was
grown i n Luria–Bertani broth containing am picillin
Table 1. Bacterial strains and plasmids use d in this study.
Strain or plasmid
Genotype or
relevant characteristic
Ref. or
source
Escherichia coli
BL21(DE3) F
–
, ompT, hsdS
B
(r
B
–
m
B
–
),
gal, dcm (DE3)
[29], Novagen
Plasmids
pCR-Blunt II
TOPO
High-copy vector for direct
cloning of PCR fragments

[28], Invitrogen
pET15b High-copy vector for
N-terminal His-tag
hyperexpression
Novagen
pET15b-ispDF Expression of ispDF from
Campylobacter jejuni as
N-terminal His-tag
fusion protein
This study
Table 2. Oligonucleotides used in this study. Restriction s ites are
underlined.Startandstopcodonsareshowninbold.
Designation Sequence (5¢-3¢)
IspDF Forward ACG
CATATGAGTGAAATGAGCCTT
ATTATGTTA
IspDF Reverse GCT
GGATCCTCATAATCTTGTCCAAT
CAAAATA
Fig. 1. Deoxyxylulose phosphate pathway for the biosynthesis of
isoprenoids.
Ó FEBS 2004 The bifunctional enzyme IspDF (Eur. J. Biochem. 271) 3029
(100 mgÆL
)1
). Cultures were incubated at 37 °Cwith
shaking. At an attenuance of 0.8 (600 n m), isopropyl thio-
b-
D
-galactoside was added to a final concentration of 1 m
M

,
and the culture was incubated at room temperature
overnight. The cells were harvested by centrifugation
(25 m in, 3500 g)at4°C.
Bacterial c ell mass ( 2 g ) was suspended in 2 0 m L of
100 m
M
Tris/HCl, pH 7 .7, containing 50 m
M
NaCl, lyso-
zyme (4 mgÆmL
)1
), benzamidine (4 mgÆmL
)1
), 1 tablet of
protease inhibitor cocktail (Roche) and 7 0 units of DNaseI
(Roche). The suspension was passed through a French press
and was then centrifuged (25 min, 39 200 g,4°C). The
supernatant was passed through a 0.2 lm fi lter and was then
appliedtoa5mLmetalchelatingHiTrapcolumn(Amer-
sham Pharmacia Biotech) preloaded with nickel chloride and
equilibrated with 100 m
M
Tris/HCl, pH 7.7, containing
50 m
M
NaCl. T he column was washed with 25 m
M
bistris
propane, pH 7 .6, containing 10 m

M
imidazole and was then
developed with a gradient of 10–1000 m
M
imidazole. Frac-
tions were analyzed by SDS/PAGE, combined and dialyzed
against 100 m
M
Tris/HCl, pH 8.5, containing 50 m
M
NaCl.
The protein concentration was determined spectrophoto-
metrically using an extinction coefficient of 2 6 000
M
)1
Æcm
)1
(280 nm). Thrombin (Amersham Pharmacia Biotech) was
added to a final concentration of 6.7 units per mg of protein.
The mixture was incubated overnight at room temperature,
passed through a 0.2 lm filter, and placed on a Sepharose Q
anion exchange column (Amersham Pharmacia Biotech)
which was developed with a gra dient of 0– 1000 m
M
NaCl.
Fractions we re combined, dialyzed against 100 m
M
Tris/
HCl, pH 8.0, and c oncentrated by ultrafiltration. Glycerol
was added t o a final concentration of 1% (v/v). The solution

was s tored at )20 °C.
NMR assay for IspD activity
A solution containing 100 m
M
Tris/HCl,pH8,10m
M
MgSO
4
,7.7m
M
[1,3,4-
13
C
3
]4,7.7m
M
CTP, 10% ( v/v)
D
2
O and protein ( total volume, 600 lL) was i ncubated in
an NMR t ube at 37 °C.
13
C NMR spectra were recorded at
intervals.
NMR assay for IspF activity
A solution containing 100 m
M
Tris/HCl, pH 8, 10 m
M
MgSO

4
,7.7m
M
[1,3,4-
13
C
3
]6,10%(v/v)D
2
O and protein
(total volume, 600 lL) was incubated in an N MR tube at
37 °C.
13
C NMR spectr a were recorded at intervals.
Fig. 2. Amino acid s equence a lignment o f t he bifunctional IspDF pro tein f rom C. jejuni and monofuntional IspD and IspF proteins from E. coli.
Residues i nvolved in substrate binding [31–33] are lab eled by triangles; conserved r esidues are shown in grey, identical residues in black.
Fig. 3. Purification o f r ecombinant IspDF p rotein from C. jejuni. (A)
molecular mass m arkers; (B) c ell extract of recombinant E. coli BL21-
pET15b-ispDF hyperexpressing the is pD F gene from C. jejuni;(C)
recombinant I spDF o f C. j ejuni protei n after n ickel c helatin g affinity
chromatography; D, recombinant IspDF after thrombin treatment
and a nion exchange chromatography on Sepharose Q .
3030 M. Gabrielsen et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Kinetic studies on recombinant IspDF protein
by NMR spectroscopy
13
C NMR signal intensities w ere used t o perform numerical
simulations using t he program
DYNAFIT
[30].

Protein sequencing
N-terminal sequencing was performed b y Edman degrada-
tion using a PE B iosystems M odel 492 (Weiterstadt,
Germany).
Mass Spectrometry
Experiments were carried out with a Voyager MALDI-TOF
mass spectrometer (Amersham Biosciencees, Inc.) using a
sinapinic acid matrix. IspDF protein from C. jejuni was
used as 0.1% (w/v) s olution in 50 m
M
Tris/HCl, pH 8.0.
NMR spectroscopy
13
C NMR spectra were r ecorded with an AVANCE 500
spectrometer from Bruker Instruments (Karlsruhe,
Germany). Composite pulse decoupling was used during
acquisition and relaxation. The s ignals of 4, 5, 6 and 7 were
assigned earlier [14–16,27]. The concentrations of the
substrates were determined by internal standardization with
sodium [1,2-
13
C
2
] acetate.
Results
A database search with the ispD and ispF genes of E. coli
identified an open reading frame of C. jejuni,acausative
agent f or gastroenteritis in humans, from the a-proteobac-
teria g roup, specifying a putative bifunctional IspDF fusion
protein (Fig. 2). Notably, however, the similarity of the

hypothetical C. jejuni gene to ispF is higher t han the
similarity to ispD. Many residues previously shown to be
part of the catalytic sites of the IspD and IspF proteins
Fig. 4.
13
C NMR signals obtained from a
reaction mixture before (A) and after incubation
(B)of[1,3,4-
13
C
3
]2C-methyl-
D
-erythritol 4-
phosphate with recombinant IspDF protein. The
mixture contained 100 m
M
Tris, pH 8.0,
7.7 m
M
CTP, 7.7 m
M
[1,3,4-
13
C
3
]2C-m ethyl-
D
-erythritol 4-phosphate and IspDF protein
from C. jejuni after nickel chelating affinit y

chromatography. The mixture was incubated
at 37 °Cand
13
C NMR spectra were recorded
at 0 min (A) and 30 min ( B).
13
C coupling
patterns o f [1,3,4-
13
C
3
]2C-methyl-
D
-erythritol
4-phosphate ( 4) and [1,3,4-
13
C
3
]4-diphospho-
cytidyl-2C-methyl-
D
-erythritol (5)areindica-
ted in (A) and ( B), respectively. In the s tructure
formulas, the
13
C labels are indicated b y filled
squares and ba rs connecting adjacent
13
Catoms.
Table 3. Activation of the catalytic domains of recombinant IspDF

protein by divalent metal ions. Th e reaction mixtures were prepared and
analyzed as described under E xperime ntal proc edure s. T he fi nal con-
centration of ea ch metal io n was 1 m
M
. The fin al concentrations of
Mg
2+
and E DTA were 10 m
M
.
Metal ion
Relative activities (%)
IspD domain IspF domain
Zn
2+
100 77
Mn
2+
96 64
Ni
2+
90 < 5
Mg
2+
90 77
Cu
2+
90 < 5
Fe
2+

87 54
Ca
2+
86 100
Co
2+
84 76
Mg
2+
82 94
EDTA < 5 < 5
Table 4. Kinetic properties of r ecombinant IspDF p rotein from Cam-
pylobacter jejuni. The reaction mixtures were pre pared and analyzed as
described under Exp erimental procedures.
Property
IspD
domain
IspF
domain
Specific activity (lmolÆmin
)1
Æmg
)1
)19 7
Turnover number (s
)1
per subunit) 13 5
K
m
(l

M
) for CTP
a
3–
K
m
(l
M
) for 2C-methyl-
D
-erythritol
4-phosphate
b
20 –
K
m
(l
M
) for 4-diphosphocytidyl-
2C-methyl-
D
-erythritol 2-phosphate
c
–19
a
Reaction mixtures containing 100 m
M
Tris/HCl, pH 8, 1 m
M
ZnCl

2
, 21.8 m
M
[1,3,4-
13
C
3
]4, 4.2 m
M
CTP, 10% (v/v) D
2
O and
0.2 l
M
protein (total volume, 600 lL) were incubated in an NMR
tube at 37 °C.
13
C NMR spectra were recorded at intervals of
8 min.
b
Reaction mixtures containing 100 m
M
Tris/HCl, pH 8,
1m
M
ZnCl
2
, 11.7 m
M
[1,3,4-

13
C
3
]4,60m
M
CTP, 10% (v/v) D
2
O
and 1.6 l
M
protein (total volume, 600 lL) were incubated in an
NMR tube at 37 °C.
13
C NMR spectra were recorded at intervals
of 8 min.
c
Reaction mixtures containing 100 m
M
Tris/HCl, pH 8,
1m
M
CaCl
2
, 19.3 m
M
[1,3,4-
13
C
3
]6, 10% (v/v) D

2
O and 3.3 l
M
protein (total volume, 600 lL) were incubated in an NMR tube at
37 °C.
13
C NMR spectra were recorded at intervals of 8 min.
Ó FEBS 2004 The bifunctional enzyme IspDF (Eur. J. Biochem. 271) 3031
[31–33], respectively, a re conserved i n t he hypothetical
C. jejuni protein (Fig. 2).
The hypothetical ispDF open r eading frame o f C. jejuni
was placed under the control o f a T
7
promoter and lac
operator in the hyperexpression plasmid pET-15b as
described under Experimental procedures. The gene was
preceded by a synthetic DNA segment specifying t he amino
acid motif MGSS(H)
6
SSGLVPRGSH designed to enable
the purification of the r ecombinant protein via metal
chelating affinity chromatography.
In a r ecombinant E. coli strain, this plasmid directed
the synthesis of a protein with an apparent mass of
about 44 kDa as judged by SDS/PAGE (Fig. 3, lane B).
Cell extracts of this strain showed catalytic activities for
IspD and IspF o f 3.9 and 0 .8 lmolÆmin
)1
Æmg
)1

, respect-
ively. The recombinant protein was purified by affinity
chromatography on a nickel c helating column (Fig. 3,
lane C).
The polyhistidine tag was removed by thrombin-medi-
ated proteolysis, and the resulting IspDF protein domain
was purified to apparent homogeneity by anion exchange
chromatography (Fig. 3, lane D). The purified protein had a
relative mass of 42 kDa as judged by polyacrylamide gel
electrophoresis. MALDI-TOF m ass spectrometry afforded
a relative mass of 41 870 Da, in close agreement with
the calculated mass o f 41 971 Da . Edman deg radation
afforded the N-terminal s equence GSHMSEMSLIM
LAAGNSTRFNTKVK where the N-terminus of the
wild-type IspDF protein was preceded by the s egment
GSH, a r emnant of the polyhistidine tag.
The catalytic act ivity of the recombinant IspDF protein
was studied by
13
C NMR spectroscopy. For reasons of
limited stability, the following kinetic assays were performed
with the polyhistidine tagged fusion protein. In order to
enhance the sensitivity and selectivity of
13
C observation,
we used m ultiply
13
C-labeled substrates. I ncubation of the
recombinant IspDF protein with [1,3,4-
13

C
3
]2C-methyl-
D
-
erythritol 4-phosphate (4)andCTPat37°C afforded
[1,3,4-
13
C
3
]4-diphosphocytidyl-2C-methy l-
D
-erythritol (5)
(Fig. 4 B; all
13
C NMR signals and
13
C
13
C coupling
constants of 4 and 5 have been reported earlier, [14]). The
optimum pH for this reaction w as pH 5. The IspD domain
was catalytically active in the presence of various divalent
metal ions (Table 3). Highe st rates of 18 .5 lmolÆmin
)1
Æmg
)1
were found with Zn
2+
.TheK

m
values for CTP a nd 4 were
3 l
M
and 20 l
M
, respectively ( Table 4 ).
Fig. 5.
13
C NM R signals o btained f rom a
reaction mixture before (A) and after incubation
(B)of[1,3,4-
13
C
3
]2C-met hyl-
D
-erythritol 4 -
phosphate with recombinant IspD F and IspE
proteins. The mixture co ntained 100 m
M
Tris
pH 8.0, 3 m
M
ATP, 9 m
M
phosphoenolpyru-
vate, 10 m
M
CTP, and 10 m

M
[1,3,4-
13
C
3
]2C-
methyl-
D
-erythritol 4-phosphate, 9 U of
pyruvate kinase, 0.2 U of IspE protein from
E. coli and IspDF from C. jejuni (total volume
600 lL). The mixture was inc ubated at 37 °C
and
13
C NMR spectra were record ed a t 0 m in
(A) a nd 3.5 h (B).
13
C c oupling patterns of
[1,3,4-
13
C
3
]2C-m ethyl-
D
-erythritol 4-phos-
phate (4) a nd [1,3,4-
13
C
3
]2C-m ethyl-

D
-erythr-
itol 2,4-cyclodiphosphate (7)areindicatedin
(A) a nd (B), respectively.
Fig. 6.
13
C NM R signals o btained f rom a
reaction mixture before (A) and after incubation
(B)of[1,3,4-
13
C
3
]4-diphosphocytidyl-2C-me-
thyl-
D
-erythritol 2-phosphate w ith r ecombinant
IspDF protein. The mixture contained 100 m
M
Tris pH 8.0, 7.7 m
M
[1,3,4-
13
C
3
]4-diphospho-
cytidyl-2C-methyl-
D
-erythritol 2-phosphate
and IspDF protein from C. jej uni after nickel
chelating affinity chrom atograph y. The mix-

ture was incubated at 37 °C.
13
CNMRspec-
tra were recorded at 0 min (A) and 30 min (B).
13
C c oupling patterns of [1,3, 4-
13
C
3
]4-
diphosphocytidyl-2C-methyl-
D
-erythritol
2-phosphate (6) and [1,3,4-
13
C
3
]2C-meth yl-
D
-erythritol 2 ,4-cyclodiphosp hate (7)are
indicatedin(A)and(B),respectively.
3032 M. Gabrielsen et al.(Eur. J. Biochem. 271) Ó FEBS 2004
The addition of ATP to the reaction mixture described
above did no t detectably affect the course of the reaction.
However, when ATP was added together with the IspE
protein of E. coli,
13
C NMR spectroscopy showed the
formation of [ 1,3,4-
13

C
3
]2C-met hyl-
D
-erythritol 2,4-cyclo-
diphosphate ( 7) as shown in F ig. 5 . T he chemical shifts as
well as the
13
C
13
C coupling constants were i n excellent
agreement with t he published data o f 7 [16].
In order to measure the rate of formation of the cyclic
diphosphate 7 from the d irect precursor, 4-diphosphocyt-
idyl-2C-methyl-
D
-erythritol 2 -phosphate ( 6), the recombin-
ant IspDF protein was incubated w ith [1,3,4-
13
C
3
]6 (Fig. 6).
The catalytic IspF domain was most active at pH 8. The
protein requires the presence of a divalent metal ion
(Table 3); highest a ctivities of 6.6 lmolÆmin
)1
Æmg
)1
(Table 4) were found with Ca
2+

.Ni
2+
and Cu
2+
could not
serve as cofactors. The K
m
value for 6 was 19 l
M
(Table 4).
A systematic a nalysis o f the genomic localization of ispD
and ispF genes in bacteria was performed using the
STRING database designed for a nalysis of f unctional
associations between proteins [34]. Data from t he genomes
of 50 bacterial species are displayed in Fig. 7. Ten eubacteria
in that collection specify bifunctional ispDF genes. In 28
species, ispD and ispF genes are directly adjacent a nd may
form part of an oper on. In all cases of fused ispD and ispF
domains as well as of directly linked ispD and ispF genes, the
ispD module is i nvariably located at the 5¢ end of t he
ensemble. Notably, all a-ande-proteobacteria studied have
fused ispDF genes s pecifying bifunctional enzymes, and all
b-andc-proteobacteria studied have adjacent genes speci-
fying monofunctional e nzymes.
Discussion
Antibiotic resistance in virtually all pathogenic bacteria now
constitutes a major public health hazard worldwide and
creates an u rgent need f or novel anti-infective strategies. A
large f raction of eubacteria including m any serious human
pathogens, for example the food-borne Campylobacter

jejuni which causes gastroenteritis [35,36], use exclusively
the nonmevalonate pathway for the biosynthesis of terpe-
noids, and the enzymes of the pathway are essential for their
survival [18,37,38]. B ecause these enzymes do not occur in
vertebrates, they are attractive therapeutic targets. It should
also be noted that the m alaria causing Plasmodium species
use the no nmevalonate pathway exclusively and can be
treated with f osmidomycin, an i nhibitor of 2 C-methyl-
D
-erythritol 4-phosphate synthase [25,26].
Clustering of ispD and ispF genes h as been reported
earlier a nd has p layed an important role in the elucidation
of the nonmevalonate pathway [14,16]. A detailed analysis
of sequence similarity and topology of the ispD and ispF
genes afforded the dendrogram i n Fig. 7 . R emarkably, all
a-ande-proteobacteria studied feature b ifunctional IspDF
enzymes. However, bifunctional enzymes are not a unique
feature of proteobacteria and are also found in the
genetically distant Spirochetes.
The putative bifunctional IspDF enzymes h ave not been
studied pr eviously in any detail. We chose the enzyme from
the human pathogen for the present s tudy with the
expectation that information on nonmevalonate pathway
enzymes from human pathogens may serve as t he basis for
antibiotic development.
Fig. 7. Phylogenetic occurrence of the ispD and ispF ge nes in d ifferent
bacterial species. Fused, linked and unlinked ispD and ispF genes are
indicated b y different a rrows.
Table 5. Catalytic properties of IspD and IspF proteins from various o rganisms. ND, not d etermined.
Organism

IspD domain IspF domain
Reference
Specific activity
(lmolÆmin
)1
Æmg
)1
)
K
m
(l
M
)
Specific activity
(lmolÆmin
)1
Æmg
)1
)
K
m
(l
M
)
Campylobacter jejuni 19 3
a
,20
b
7 19 This study
Escherichia coli 23 3

a
, 131
b
2.5 ND [39,40]
Arabidopsis thaliana 67 114
a
, 500
b
ND ND [39]
Plasmodium falciparum ND ND 4 252 [41]
a
Determined for CTP.
b
Determined for 2C-methyl-
D
-erythritol 4-phosphate.
Ó FEBS 2004 The bifunctional enzyme IspDF (Eur. J. Biochem. 271) 3033
The catalytic p roperties of the IspD and IspF domains of
the bifunctional C. jejuni enzyme are similar to those of the
orthologous, monofunctional enzymes from various organ-
isms (Table 5) [38–40]. Whereas the IspF enzymes of E. coli
and Plasmodium falcip arum catalyze a side reaction condu-
cive to the formation of 2C-methyl-
D
-erythritol-3,4- cyclo-
monophosphate [16,40], the C. jejuni enzyme exclusively
forms 2C-methyl-
D
-erythritol 2,4-cyclodiphosphate ( 7).
Acknowledgements

We thank Jane Turner a nd Brendan Wren for supplying the
genomic DNA of C. jejuni and Angelika Werner and Fritz W endling
for expert h elp with the pre paration of the manuscript. This work was
supported by the Wellcome Trust, the Deutsc he Forschungsgemeinsc-
haft, the Fonds der Chemischen Industrie and the Hans-Fischer-
Gesellschaft.
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