doi:10.1016/j.jmb.2004.10.030 J. Mol. Biol.

(2005) 345, 115–127

The Crystal Structure of E. coli 1-Deoxy-D-xylulose-5-

phosphate Reductoisomerase in a Ternary Complex
with the Antimalarial Compound Fosmidomycin and
NADPH Reveals a Tight-binding Closed Enzyme
Conformation
Aengus Mac Sweeney1†, Roland Lange1†, Roberta P. M. Fernandes2
Henk Schulz1, Glenn E. Dale1, Alice Douangamath1, Philip J. Proteau2
and Christian Oefner1*
1
Morphochem AG The key enzyme in the non-mevalonate pathway of isoprenoid biosyn-
WRO-1055/338 thesis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) has been
Schwarzwaldallee 215 shown to be the target enzyme of fosmidomycin, an antimalarial,
CH-4058, Basel, Switzerland antibacterial and herbicidal compound. Here we report the crystal
2 structure of selenomethionine-labelled Escherichia coli DXR in a ternary
College of Pharmacy, Oregon complex with NADPH and fosmidomycin at 2.2 Å resolution. The
State University, Corvallis
structure reveals a considerable conformational rearrangement upon
OR 97331-3507, USA
fosmidomycin binding and provides insights into the slow, tight binding
inhibition mode of the inhibitor. Although the inhibitor displays an
unusual non-metal mediated mode of inhibition, which is an artefact most
likely due to the low metal affinity of DXR at the pH used for
crystallization, the structural data add valuable information for the rational
design of novel DXR inhibitors. Using this structure together with the
published structural data and the 1.9 Å crystal structure of DXR in a
ternary complex with NADPH and the substrate 1-deoxy-D-xylulose 5-
phosphate, a model for the physiologically relevant tight-binding mode of
inhibition is proposed. The structure of the substrate complex must be
interpreted with caution due to the presence of a second diastereomer in
the active site.
q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author Keywords: DXR; IspC; crystal structure; fosmidomycin; malaria

† A.M.S. & R.L. contributed equally.
Present addresses: R. Lange, Actelion-Percurex AG,
Schwarzwaldallee 215, 4058 Basel, Switzerland; A.
Douangamath, Evotec OAI, 151 Milton Park, Abingdon,
Oxon OX14 4SD, UK.
Abbreviations used: DTT, dithiothreitol; DXP, 1-D-
deoxyxylulose-5-phosphate; DXR, DXP
reductoisomerase; fosmidomycin/FR-31564, 3-(N-
hydroxy)aminopropylphosphonic acid; NADPH,
nicotinamide adenine dinucleotide phosphate; NCS, non-
crystallographic symmetry; NTA, nitrilotriacetic acid
agarose; SeMet, selenomethionine; TCEP, Tris(2-
carboxyethyl)-phosphine; TIM, triose phosphate
isomerase.
E-mail address of the corresponding author:
christian.oefner@morphochem.ch
Introduction
Malaria remains a significant cause of mortality
in many areas of the world, with over 300 million
cases and one million deaths per year.1 The
escalating incidence of malaria and the develop-
ment of drug resistance highlights the urgent need
to identify novel antimalarial target enzymes and
drugs. Recently two phosphonic acid antibiotics,
fosmidomycin (FR-31564) and its derivative
FR-900098 have been identified as promising agents
for the development of a new antimalarial chemo-
therapy.2,3 Originally, these compounds were
identified as spheroplast-inducing antibacterials
inhibiting most Gram-negative and some Gram-
positive bacteria.4 Initial studies undertaken to
illuminate the mode of action indicated that

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
116
fosmidomycin blocks bacterial isoprenoid bio-
synthesis. 5 Subsequently, it was shown that
fosmidomycin and FR-900098 (Figure 1) are specific
inhibitors of 1-deoxyxylulose-5-phosphate reducto-
isomerase (DXR; EC 1.1.1.267) also known as 2-C-
methyl-D-erythritol synthase (MEP synthase) and
IspC.2,6,7 DXR is the second enzyme of the
mevalonate-independent pathway for the bio-
synthesis of isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP), the two
basic precursors for isoprenoids. 6 Genomic
analyses of the phylogenetic distribution indicate
that the genes coding for this mevalonate-indepen-
dent pathway are present in most Gram-negative
and some Gram-positive bacteria, in higher plants
and Plasmodium falciparum but not in mammals,
fungi and archaebacteria.8,9 Accordingly, the phylo-
genetic distribution of dxr (yaeM) is consistent with
the activity spectrum of fosmidomycin as an
antibacterial, antimalarial and herbicidal agent.
The enzymatic properties of DXR and its mode of
inhibition by fosmidomycin have been analyzed in
detail. DXR catalyzes the conversion of 1-deoxy-D-
xylulose-5-phosphate (DXP) to 2-C-methyl-D-ery-
thritol phosphate (MEP), requiring NADPH and a
divalent metal such as Mn2C, Mg2C or Co2C (Figure
2).6,10,11 Based on the stereochemical features of the
NADPH-dependent reduction, DXR is classified as
a class B dehydrogenase.12,13 Recent mechanistic
investigations suggest a two-step reaction involving
an isomerization of DXP to 2-C-methyl-D-erythrose-
4-phosphate followed by NADPH-dependent
reduction to form 2-C-methyl-D-erythritol-4-phos-
phate. Two possible mechanisms have been pro-
posed for isomerization by DXR, an alpha-ketol
rearrangement and a retroaldolization/aldolization
(Figure 2).14 The divalent ion is required for the
reduction step but whether it is required for the
initial isomerization step has not been determined.
The catalytic role of His209 has also not been
determined but it is likely that it plays a role in
binding the substrate in the correct orientation and
conformation. It has been shown that mutation
of His209 to Gln causes a 5200-fold decrease in
kcat/Km.10
An analysis of the progress curves for MEP
synthesis in the presence of inhibitor revealed a
slow, tight-binding mode of inhibition by fosmido-
mycin and two kinetically distinguishable Ki values
have been reported for the Escherichia coli enzyme
(Ki w215 nM, Ki
w21 nM).11 As the slow onset of
Figure 1. The DXR inhibitors fosmidomycin (A) and
FR-900098 (B).
Crystal Structure of DXR
inhibition is eliminated when the enzyme DXR is
preincubated with fosmidomycin and NADPH but
not when NADPH is omitted from the preincu-
bation mix it has been concluded that the tight
binding of the inhibitor requires prior formation of
a DXR–NADPH complex. A slow onset of inhi-
bition, as displayed by fosmidomycin with DXR, is
typically associated with a conformational change
of the enzyme.
Four crystal structures of DXR from E. coli have
been determined to high and medium resolution as
the enzyme alone (1K5H),15 a binary complex with
NADPH (1JVS),16 a complex with fosmidomycin
and manganese (1ONP)17 and a complex with
bisphosphonate inhibitors and manganese.18 The
crystal structure of DXR from Zymomonas mobilis, a
bacterium widely used for ethanol production, has
recently been reported as an apoenzyme (1ROK)
and a binary complex with NADPH (1ROL).19 The
V-shaped protein displays an intrinsic flexibility15,16
and it has been proposed that binding of mag-
nesium, substrate or inhibitor could induce a
conformational change. Conformational differences
between the three molecules of the asymmetric unit
of the apoenzyme, resulting from different crystal
contacts, revealed that the connective domain
(residues 160–270) can function as a hinge. High
temperature factors (approx. 100 Å2) in the pub-
lished structures, with the exception of molecule C
of the apoenzyme (PDB code 1K5H)15 which is
stabilized by crystal contacts, reveal disorder in
loop 206–216. However, the structure of DXR
complexed with fosmidomycin in the presence of
manganese through soaking experiments17 reveals
no structural rearrangement and little order in loop
206–216, suggesting that the observed complex
might represent the initial, lower affinity (210 nM)
binding state of fosmidomycin. Published data have
shown that slow, tight binding inhibition requires a
conformational change, which occurs only upon
prior binding of NADPH.11
Here we report the structure of both wild-type
and SeMet-labelled DXR determined from crystals
cocrystallized in the presence of the inhibitor
fosmidomycin, magnesium and the required cosub-
strate NADPH. The enzyme undergoes the
expected rearrangement upon complex formation
and the structure reveals a binding mode of
fosmidomycin that is not mediated by a metal ion.
The ternary complex of the DXR, NADPH and the
substrate DXP is also presented and is used to create
a model of metal-mediated fosmidomycin binding.
Results
Overall structure
The overall structure of the complexed enzyme is
similar to that described,15–19 with a symmetry-
related molecule forming the physiologically active
homodimer in the case of the fosmidomycin
complexes. The structure is composed of three
Crystal Structure of DXR
domains: a larger N-terminal NADPH binding
domain comprising 150 residues, a connective or
linker domain (residues 160–270) and a smaller
C-terminal alpha-helical domain comprising resi-
dues 312 to 398 (Figure 3). The N-terminal domain
exhibits an a/b topology with a seven-stranded
parallel b-sheet and seven a-helices. The connective
domain comprises five helices and a four-stranded
b-sheet with one parallel and two antiparallel
alignments. The open side of the b-sheet, i.e. the
side that is not covered by loops or helices, forms
part of the protein surface. The C-terminal domain
is a four-helix bundle and is joined to the connective
domain by an extended loop (residues 186–216),
which spans the entire central domain and contains
eight conserved residues. As is the fold, the dimer
interface is identical to that observed in the non-
crystallographic symmetry (NCS)-related dimers of
the known structures. With the exception of the
N-terminal six-histidine tag, all residues of the
protein are well ordered. The structure of DXR in
complex with NADPH (PDB accession code 1JVS,
molecule A) was used for comparison. In the
structure of the substrate complex, two crystal-
lographically independent monomers were
117
Figure 2. The conversion of DXP
to MEP via the intermediate 2-C-
methyl- D -erythrose-4-phosphate.
The carbon numbering scheme for
DXP is shown.
observed which revealed almost identical confor-
mations despite differences in crystal packing.
The most notable difference between the pub-
lished structures of DXR and the fosmidomycin and
substrate ternary complexes observed in the pre-
sence of NADPH is an induced fit adaptation of the
connective and C-terminal domains upon small
molecule binding, resulting in a closed enzyme
conformation. Superimposing the NADPH binding
domain of the fosmidomycin complex structure
with that of the binary complex reveals a 12.58
domain rotation of the remaining domains (Figure
3). Residues 206–216 form a flexible “lid”, which
closes upon substrate or fosmidomycin binding to
shield the active site from the solvent. The ternary
complex structures reveal for the first time a fully
ordered conformation of loop 206–216 that is not
dependent on crystal contacts. Gly185 appears to
act as a hinge, with Ser186 forming a hydrogen
bond with His209 in the closed enzyme confor-
mation. The dihedral angle phi of G185 is altered by
approximately 358 between the open (binary
NADPH complex) and closed conformations of
DXR. A comparison of the open and closed enzyme
conformations using the conformational analysis
118
Figure 3. The physiological homodimer of DXR. The
lower monomer is colored yellow (N-terminal domain),
green (connective domain), blue (C-terminal domain) and
grey (flexible “lid” loop). The upper monomer is colored
dark blue with the “lid” loop shown in red. The upper
monomer has been superimposed with the binary
complex of NADPH with DXR by fitting the N-terminal
domains (residues 1–153) and the regions of binary
complex showing the greatest structural difference
(residues 154–230 and 298–398, average Ca deviation
3.2 Å each domain) are shown in magenta. The average
Ca deviation of the omitted regions is 0.8 Å.
software DynDom20 indicates a domain movement
upon inhibitor binding, involving the region of
Gly185 as a hinge for the structural alteration.
The binding of the adenine and pentose phos-
phate moieties of NADPH is identical to that
observed in the structure of the binary complex.16
The nicotinamide ring of NADPH is disordered in
the fosmidomycin complexes but not in the sub-
strate complex. The NADPH molecule in the SeMet-
labelled DXR–fosmidomycin complex structure
turns away from the active site and is disordered
from the nicotinamide ring to the nearest phosphate
link. This appears to be caused by the conformation
of SeMet214, which occludes the binding pocket of
the nicotinamide ring. In the wild-type DXR–
fosmidomycin and DXR–substrate complexes
NADPH can be clearly seen to extend to the active
site, although the nicotinamide moiety of the
fosmidomycin complex remains partially
Crystal Structure of DXR
disordered. The entire NADPH molecule is clearly
visible in the substrate structure. In this NADPH
conformation, two additional hydrogen bonds are
formed with Gly215(N) compared to the NADPH
binary complex. The favorable hydrogen bonding
of NADPH in the closed conformation may account
for its mandatory binding prior to the formation of
the high affinity DXR–fosmidomycin complex.
Interestingly, the published DXR–NADPH complex
exhibits a partially closed conformation that is
between the most open structure of DXR alone
and the closed structure of the DXR-NADPH–
fosmidomycin complex described here (Figure 4).
In order to investigate the driving force for
“closure” of the enzyme, the solvent-accessible
surface areas of the ternary complexes and the
published NADPH binary complex16 were com-
pared. Upon adopting the closed conformation, the
hydrophobic surface area of DXR is decreased by
4.1% (710 Å2) while the polar surface area is
increased by 2.9% (400 Å2). Both molecules of the
dimer were used in all surface area calculations,
and similar values were obtained for the substrate
and fosmidomycin ternary complexes. The solvent-
exposed surfaces of the hydrophobic residues
Trp212, Ile218 and Met276 in particular are signifi-
cantly reduced upon inhibitor binding.
Active site
Within the active site region, the inhibitor and
substrate bind to both the connective and C-term-
inal domains and appear to induce the observed
conformational change by tethering the domains in
a closed conformation. In addition, the flexible loop
connecting both domains (residues 206–216) under-
goes an induced fit adaptation and adopts a
conformation that allows it to function as a “lid”
over the active site. This significant rearrangement
points to its important role in substrate binding. The
highly conserved residues Met276, Met214 and
Figure 4. This orientation shows DXR with nothing
bound (green), NADPH bound (yellow) and NADPH and
fosmidomycin bound (blue).
Crystal Structure of DXR
Trp212 form a barrier between the active site and
the solvent and the b-indole of Trp212 provides the
key interaction with the substrate or fosmidomycin
backbone. This arrangement results in the for-
mation of an intramolecular interface of 160 Å2, of
which about 80% is of hydrophobic character. The
most notable effect of the conformational change is
the shielding of the active site region from the
solvent. The electron density for NADPH and loop
206–216 of the substrate complex are shown in
Figure 5.
The substrate-binding cavity can be considered to
consist of three regions: a positively charged pocket,
which binds the phosphonate moiety of fosmido-
mycin, a hydrophobic region around the carbon
backbone and an amphipathic region, which binds
the hydroxamic acid function. The active site
contains the highly conserved residues Asp150,
Glu152, Glu231, Glu234, His209 and His257. The
clustered acidic Asp and Glu residues are thought
to bind the catalytically essential divalent cation, by
analogy with the structure of acetohydroxy acid
isomeroreductase.21 DXR and acetohydroxy acid
isomeroreductase catalyze similar reactions
although the two enzymes are unrelated by
sequence or structure. The acidic residues at the
active site of the NADPH binary complex interact
with the highly conserved basic residues Lys125,
His153 and Lys228 and the formation of a complex
with the substrate or fosmidomycin causes sub-
stantial changes in the hydrogen bonding network
of these residues. The proximity of the carboxylates
of Asp150, Glu152 and Glu231 suggests that they
interact via hydrogen bonds and raises uncertainty
concerning their protonation states.15 In the closed
conformation of the enzyme, the newly created
hydrophobic nature of the active site may alter the
pKa of these residues further, as is observed for the
catalytic base Glu165 of triose phosphate isomerase
(TIM).22
119
Fosmidomycin binding
Within the solvent-shielded cavity that is formed
upon closure of the “lid” residues 206–216, the
inhibitor backbone lies parallel to the b-indole of
Trp212 at a distance of approximately 4 Å. The
hydroxamic acid moiety of the inhibitor binds to the
side-chains of Glu152 and Glu231 and to the
backbone nitrogen of Ser151 (Figure 6). A change
of 1208 in the chi-1 torsion angles of both Asp150
and Glu234 residues results in the exchange of the
hydrogen bond between Asp150 and Glu152 (6.0 Å
versus 2.8 Å in the published binary complex with
NADPH) with a bond between Asp150 and Glu234
(3.0 Å versus 5.0 Å in the binary complex). Remark-
ably, no magnesium ion was observed in either this
structure or those of the apoenzyme and binary
complex, despite the fact that it is essential for
activity and was included in the crystallization
experiments. Furthermore, the clustered acidic
residues of the active site do not give rise to
octahedral geometry, which is required for mag-
nesium binding. This is most likely due to the low
pH of the crystallization buffer (pH 5.0), which
reduces the metal-binding affinity of DXR. A
schematic diagram of fosmidomycin binding to
SeMet-labelled DXR is shown in Figure 6(C).
Upon superimposing the hydroxamate-binding
region of the fosmidomycin complex with the
corresponding region of the NADPH binary com-
plex, the phosphonate moiety of the inhibitor is
observed to lie 1.9 Å from the position occupied by
the sulphate ion in the binary complex. The
phosphonate-binding region of DXR is also dis-
placed by 1.9 Å toward the hydroxamate binding
region, in a movement that matches that of the
overall domain. Fosmidomycin forms no hydrogen
bond with His209, which appears to plays an
important role in binding the substrate in the
correct orientation for catalysis (Figure 7).10 This
may be due to the presence of a monoanionic
Figure 5. An FoKFc omit electron
density map of NADPH and loop
206–216 of the substrate–NADPH–
DXR ternary complex, contoured at
3s.
120 Crystal Structure of DXR

Figure 6. (A) An FoKFc omit electron density map of fosmidomycin in the active site of DXR, contoured at 3s. Met276
is shown in orange on the left and Met214 is shown in two conformations: that of SeMet214 in magenta (right) and that of
wild-type Met214 in orange (left). (B) Hydrogen bonding interactions of the inhibitor fosmidomycin in the active site of
DXR. (C) A schematic diagram of fosmidomycin binding to SeMet-labelled DXR.

phosphonate species of fosmidomycin resulting fosmidomycin. The more open inhibitor-binding

from crystallization at pH 5.0, instead of the
dianionic species that exists at a physiological pH.
Interestingly, Met214 of the selenomethionine-
labelled fosmidomycin complex occupies a differ-
ent position to that observed in the wild type
complex structure, with atoms Se and CE lying
closer (3.0 and 6.1 Å) to the corresponding atoms of
Met276 (Figure 6(A)). In this conformation, Met214
forms a hydrophobic interaction with Met276 rather
than the inhibitor backbone. This conformational
change can be explained by the fact that seleno-
methionine is more hydrophobic than methionine,
with a difference of 0.17 kcal/mol in their free
energies of transfer from octanol to water.23 As can
be seen from the omit map (Figure 6(A)), SeMet214
partially occupies both possible conformations
although the conformation shown in Figure 6(B) is
favored. Whether the conformational difference
between Met214 and SeMet214 reflects a flexibility
of Met214 that is required for substrate binding
remains unclear. In the SeMet-labelled enzyme a
closed cavity of 713 Å3 is formed upon inhibitor
binding, of which 464 Å3 is occupied by
pocket of the wild-type DXR–fosmidomycin com-
plex remains largely shielded from the solvent but
merges with the binding pocket of the nicotinamide
ring of NADPH. Overall, the two protein structures
are almost identical in all other respects.
Substrate binding
The phosphate moiety of the substrate forms
hydrogen bonds with Ser186, Asn227, Lys228 and
with the catalytically essential His209 (Figure 7).
These residues are hydrogen bonded to a sulphate
ion in the apoenzyme and NADPH binary complex
structures. The substrate backbone interacts with
the b-indole of Trp212 and the carbonyl group of C2
is hydrogen bonded to Glu152 and the backbone
nitrogen of Ser151. The hydroxyl group of C3 is
involved in hydrogen bonds with Lys125 and
Glu231, while the C4 hydroxyl group is hydrogen
bonded to Glu152, Asn227 and Lys228. The C3
hydroxyl group of the alternative diastereomer is
not involved in any hydrogen bonding interactions.
The nicotinamide ring of NADPH adopts an
Crystal Structure of DXR 121

Figure 7. (A) An FoKFc omit electron density map of the substrate DXP in the active site of DXR, contoured at 3s. The
position of the hydroxyl group in the L-configuration at C4 is depicted in green. (B) Hydrogen bonding interactions of
the substrate DXP in the active site of DXR. The position of the hydroxyl group in the L-configuration at C4 is depicted in
green. (C) A schematic diagram of the substrate DXP binding to DXR. For clarity, the bond between the carboxylate of
E231 and the hydroxyl group of C4 of DXP has been omitted.

orientation that would allow the transfer of a Model of tight-binding mode of fosmidomycin
hydride from the pro-S hydrogen of C4 of the
nicotinamide ring to C2 of the proposed intermedi- A model of metal-mediated fosmidomycin inhi-
ate 2-C-methylerythrose 4-phosphate, which is in bition of the closed form of DXR (shown in Figure 8)
keeping with the classification of DXR as a class B
dehydrogenase.12 A schematic diagram of DXP
binding to DXR is shown in Figure 7(C).
The electron density indicates that two diaster-
eomers are bound to the enzyme, although NMR
analysis of the substrate indicated the presence of a
single diastereomer. For this reason, it was not
possible to unambiguously interpret this crystal
structure with regard to the enzyme mechanism.
However, because the structure was determined at
a physiologically relevant pH, it has been possible
to use both available structures to create a model
for the tight-binding inhibitor of fosmidomycin.
The reason for the presence of a second dia-
stereomer is unclear and may be due to epimeriza-
tion under the crystallization conditions or the
selective binding of a small amount of the non- Figure 8. A model of metal-mediated fosmidomycin
physiological diastereomer by metal-free DXR. binding to DXR in its tight-binding conformation.
122
was prepared based on the structure on the ternary
complexes and the published structure of fosmido-
mycin bound to DXR in its open conformation.17
The phosphonate moiety of fosmidomycin is bound
in a similar fashion to the phosphate group of the
substrate. The hydroxamic acid moiety coordinates
a divalent cation that is bound by residues D150,
E152 and E231, the same residues that coordinate
the metal in the published structure of the binary
complex of DXR and fosmidomycin.17 The metal
binding site was modelled using the octahedral
binding site that was observed in the metal-free
substrate complex as well as the observed matching
position of the metal in the published fosmidomy-
cin binary complex. The inhibitor was adjusted
slightly to accommodate the metal ion. The inter-
actions of the metal with the inhibitor and the
enzyme are similar to those observed in the
published structure of fosmidomycin bound to
DXR. As in the published structure a single water
molecule completes the octahedral coordination of
the metal, however in the model this water
molecule is also hydrogen bonded to K125. The
favorable hydrophobic interactions of the inhibitor
with Trp212, Met214 and Met276 that were
observed in the structure of the ternary complex
of fosmidomycin, NADPH and DXR are preserved
in this model. By contrast, these interactions are not
observed in the published structure of the binary
complex of fosmidomycin and DXR. Met276 and
the nicotinamide ring of NADPH also contribute to
the formation of a hydrophobic binding pocket. The
tight-binding conformation of the enzyme results in
a small and totally enclosed inhibitor-binding
pocket. This model is consistent with the fact that
no inhibitors of DXR that are significantly larger
than fosmidomycin have been reported to date. It
also reveals that the only possibility to extend the
molecule will involve either displacement of the
nicotinamide ring of NADPH or targeting the open
conformation of DXR. We propose that this model
represents the physiologically relevant tight-bind-
ing conformation of DXR in complex with
fosmidomycin.
Kinetics
The Michaelis constants (Km) for the divalent
cations Mg2C, Mn2C and Co2C with E. coli DXR
were determined at two different pHs, pH 7.8 and
pH 6.0. The results are summarized in Table 1. An
Table 1. Michaelis constants for divalent cations using
E. coli DXR
Km (mM)
Cations pH 7.8 pH 6.0
Mn 2C
17G2 520G60
Mg2C 210G20 17,000G2000
Co2C 2.0G0.7 nd
nd, not determined.
Crystal Structure of DXR
attempt was made to determine the Kms at pH 5.0,
but the enzyme activity was negligible in either
citrate or acetate buffer at this pH. The Kms
determined at pH 7.8 for the ions Co2C and Mn2C
are 2 mM and 17 mM, respectively, similar to the Kms
found for these ions with the DXR from Synecho-
cystis (Co2C 10 mM; Mn2C 15 mM)23 and Mycobacter-
ium tuberculosis (Co2C 1.2 mM; Mn2C 21 mM).24 The
Km value observed for Mg2C was 210 mM. Although
this value is higher than those determined for Mn2C
and Co2C, it is lower than the values found with the
Synechocystis (2400 mM; 11-fold) and Mycobacterium
tuberculosis (1200 mM; sixfold) DXRs.24,25 Mg2C has
been proposed to be the physiologically relevant
metal ion activator for DXR.11
An increase in the Km values for Mn2C and Mg2C
was observed when the assays were carried out at
pH 6.0 (Table 1). The Km for Mn2C increased 31-fold
and for Mg2C 81-fold. A similar pH effect was
observed using the M. tuberculosis DXR, where the
Km for Mn2C increased 243-fold from 7 mM at pH
8.75 to 1.7 mM at pH 6.0.24 The increase in Km is
most likely due to a change in protonation state of
the carboxylate ligand(s) for the divalent metal
ion.24 The carboxylic acid functional group is a
poorer ligand than a carboxylate. Due to the low pH
of the crystallization buffer and a concentration of
10 mM Mg2C, it appears as though the absence of a
Mg2C in the crystal structure results from a
decreased affinity for the divalent ion at low pH.
Discussion
The structures of both ternary complexes reveal a
dramatic conformational change of DXR that is
induced by substrate or inhibitor binding in the
presence of NADPH. This remarkable flexibility of
DXR plays an important role in substrate binding
and catalysis and is similar to that of other
isomerases in which a domain or loop movement
is required to shield a reactive intermediate from
the surrounding solvent.22,26 Based on the slow
timescale of DXR domain movement compared to
the rate of inhibitor diffusion, it is possible to
explain the slow onset of inhibition that has been
observed for fosmidomycin. The formation of
additional hydrogen bonds between the enzyme
and NADPH in the closed conformation correlates
with the observation that NADPH is essential for
tight binding of the inhibitor.11 In addition, the
favorable enthalpic interactions due to the
increased buried hydrophobic surface in the closed
conformation may compensate for the entropic cost
of a more rigid closed structure. The increase in
buried hydrophobic surface area in the closed
conformation, together with its occurrence under
different crystal packing conditions obtained by
cocrystallization experiments indicates that the
observed conformation most likely represents that
of the complex in solution.
The structure of the ternary complex of DXR with
NADPH and fosmidomycin differs markedly from
Crystal Structure of DXR
that published by Steinbacher et al.,17 in which the
hydroxamic acid of fosmidomycin co-ordinates a
manganese ion which is bound to Asp150, Glu152
and Glu234. In the previously published structure,
the octahedral geometry of the manganese-binding
site is completed by three water molecules and the
active site lies on the enzyme surface due to the
open conformation of the enzyme. No hydrophobic
interactions were observed between the methylene
linker of fosmidomycin and the flexible “lid” loop of
DXR (residues 206–216) is disordered. By contrast,
in the tight-binding conformation fosmidomycin is
buried within a solvent-shielded binding site. The
active sites of both structures are shown in Figure 9,
in which both fosmidomycin molecules have been
superimposed. The greatest difference between the
two active sites is observed in loop 206–216, which
is largely disordered in the previously published
structure of fosmidomycin bound to DXR. In
addition, the region containing Met276 is closer to
fosmidomycin in the ternary complex than in the
previously published structure. The absence of the
expected metal ion from the structure can be
explained by the low metal affinity of DXR in the
crystallization buffer. The fact that the same enzyme
conformation was observed in both ternary com-
plexes, despite different crystallization conditions
and crystal packing, allows us to conclude that the
structures reveal the physiologically relevant sub-
strate/inhibitor binding conformation of DXR. The
previously solved structure of DXR in an open
conformation in complex with fosmidomycin17 may
represent the initial, lower affinity enzyme–inhibitor
complex.
The ternary complex of DXR, NADPH and
substrate reveals significant differences between
the binding modes of the inhibitor and substrate,
even in their phosph(on)ate moieties. However,
because the electron density indicates the presence
123
of a second diastereomer (at C-3), it is impossible to
draw any firm conclusion about the enzyme
mechanism based on this structure. Two features
of the substrate-binding mode are noteworthy.
Firstly, the catalytic residue His209 is hydrogen
bonded to the phosphate moiety of the substrate,
which is separated by four atoms (a distance of
5.1 Å) from the scissile carbon-carbon bond. Thus, it
is tempting to speculate that the role of the histidine
residue might involve “pre-orientation” of the
substrate in an orientation and conformation that
favors retroaldolization or a-ketol rearrangement.
The role of phosphate binding in stabilizing
conformations that favor productive turnover has
been reported for the enzyme ribulose-bisphos-
phate carboxylase (RuBisCo).27 However, further
biochemical and structural studies will be required
to determine the exact role of the histidine residue.
Secondly, a distorted octahedral coordination site
involving Glu152, Glu231, Asp150, Lys125 and the
carbonyl group of C2 and the hydroxyl group of C3
of the substrate appears to be the most likely
magnesium-binding site. This contrasts with the
fosmidomycin complex in which no metal-binding
site was observed.
In summary, our findings reveal the closed
conformation of DXR and allow us to propose a
model for the tight-binding mode of fosmidomycin,
which will be of use for the development of novel
DXR inhibitors.
Materials and Methods
Cloning, overexpression and purification
The E. coli dxr (ispC) gene (yaeM, accession number
AE000126) encoding 1-deoxy-D-xylulose-5-phosphate
reductoisomerase (EC 1.1.1.267) was amplified by
Figure 9. The superimposed
active sites of the published DXR-
fosmidomycin complex and the
ternary complex of fosmidomycin,
NADPH and DXR. The previously
published complex is shown in
yellow and the ternary complex is
shown in grey.
124
polymerase chain reaction (PCR) from genomic E. coli
K12 (W3110) DNA using the oligonucleotide primers
Ecdxrfor (5 0 -GGATGTCATATGAAGCAACTCACCATTC)
and Ecdxrrev (5 0 -GACTATATCACTGGATCCCTACGC
AAC). The respective PCR product was purified, digested
with NdeI and BamHI and inserted into the NdeI-BamHI
digested vector pDS-6hisNdeI (27). Insertion into pDS-
NdeI6his resulted in a recombinant dxr gene with a 5 0 -end
coding for an N-terminal tag of additional 14 residues
including six consecutive histidine residues (H6-DXR).
The dxr nucleotide sequence in pDS-NdeI-6hisEcdxr was
verified by applying the dye terminator cycle sequencing
technology on an ABI PRISM 310 Genetic Analyzer. For
routine expression of H6-DXR the E. coli host strain BL21
was first transformed with pREP4 (lacIq, Qiagen) and
then with pDS-NdeI-6hisEcdxr. Expression of H6-DXR
was induced by the addition of 0.25 mM isopropyl-b-D-
thiogalactopyranoside to mid-log cells growing in nutri-
ent rich medium (NZ-amine 1%, Yeast extract, NaCl)
supplemented with 0.2% (w/v) fructose, 100 mg/ml
ampicillin and 25 mg/ml kanamycin at 30 8C. Cells were
grown for an additional 2.5 hours and then harvested by
centrifugation.
In vivo incorporation of SeMet into H6-DXR was
achieved by applying the methionine pathway inhibition
method.28,29 BL21 pREP4 pDS-NdeI-his6-Ecdxr was
grown overnight in minimal medium M9 (30) sup-
plemented with 0.2% fructose, 1 mg/ml thiamin, 25 mg/ml
kanamycin and 100 mg/ml ampicillin at 30 8C. Two liters
of the same medium were inoculated with cells from the
overnight culture to reach an absorbance of A600 nmZ
0.12). Cells were harvested after 4.5 hours growth,
centrifuged and resuspended in two liters of fresh,
prewarmed (30 8C) minimal medium containing
additional amino acids (L-lysine, L-phenylalanine, L-
threonine at 100 mg/l; L-isoleucine, L-leucine, L-valine at
50 mg/l). After 15 minutes growth in the presence of
these amino acids expression of H6-DXR was induced by
adding 0.5 mM IPTG. After two hours cells were
harvested (6000 g, 15 minutes) and stored at K80 8C.
H6-DXR protein has been purified by a combination of
Ni-NTA affinity chromatography and size exclusion
chromatography. Cells were resuspended in ice-cold
lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM
imidazole 5 mM TCEP pH 8) containing Protease Inhibi-
tor Cocktail (Roche). After incubation in the presence of
1 mg/ml lysozyme cells were broken by ultrasonic
treatment. Cell debris were removed by centrifugation
(20,000 g, 30 minutes, 4 8C). The bacterial lysate was
mixed with 50% Ni2C-NTA (Qiagen) and incubated for
one hour at 4 8C. The lysate-Ni2C-NTA mixture was
loaded onto an Econo Pac column (Biorad) and washed
with 12 volumes of wash buffer (NaH2PO4, 300 mM NaCl,
20 mM imidazole, pH 8). H6-DXR was eluted with buffer
(NaH2PO4, 300 mM NaCl, 250 mM imidazole). H6-DXR
containing fractions were identified by SDS-PAGE,
pooled and concentrated by ultrafiltration using ULTRA-
FREE-15 filter devices (Millipore, MWCO 30,000). An
aliquot of 10 ml of the H6-DXR containing solution was
purified on a Superdex 75 gel filtration column equili-
brated with 50 mM Tris, 100 mM NaCl, 2 mM MgCl2,
5 mM TCEP, 5% (v/v) glycerol. H6-DXR eluted as a single
peak. For the preparation of metal-free H6-DXR, the metal
(MgCl2) was omitted from all buffers and 20 mM EDTA
was added to the buffer used for size exclusion
chromatography. Protein concentrations of pure fractions
were determined by absorbance at 280 nm. Enzymatic
activity of H6-DXR was routinely determined at 30 8C in
40 mM Tris–HCl buffer containing 2 mM MgCl2 or 2 mM
Crystal Structure of DXR
MnCl2, 2 mM DTT, 1 mg/ml BSA buffer at pH 8 contain-
ing varying concentrations of NADPH and DXP. The
enzymatic reaction rate was measured by monitoring the
changes at A340 nm corresponding to the oxidation of
NADPH (30340 Z 6:22 cmK1 mMK1). The substrate (DXP)
was synthesized by the method described by Blagg &
Poulter.30
Kinetic studies
The typical assay system for kinetic studies contained
100 mM Tris–HCl (pH 7.8), 1 mM MnCl2, 2 mM DXP,
0.2 mM NADPH, and 1 mg/ml of BSA in a final volume
of 1 ml. Assays were conducted at pH 6.0 used 100 mM
Mes buffer. The reaction was initiated by adding the
enzyme (5 nM at pH 7.8; 50 nM at pH 6.0) to the total
assay mixture. The oxidation of NADPH was monitored
at 37 8C in a Hitachi U-2000 Spectrophotometer. The
kinetic parameters for three metal ions, Mn2C, Mg2C and
Co2C, were determined at pH 7.8 and 6.0. Kinetic
parameters were determined in triplicate using at least
seven varying concentrations of these ions. The concen-
tration ranges for Mn2C, Mg2C and Co2C were 2–
2,000 mM, 50–60,000 mM and 0.4–500 mM, respectively.
Kinetic parameters were calculated by fitting the initial
velocity versus substrate concentration to the Michaelis
equation, vZ ðVmax ½SÞ=ðKm C ½SÞ, using the non-linear
regression analysis option of GraphPad Prism software.
Crystallization
Crystallization screening was carried out by the
modified microbatch method,31 adding 1ul of protein to
1ul of crystallization solution from the INDEX screen
(Hampton Research catalogue No. HR2-134). A Douglas
Instruments Impax robot was used for pipetting. Crystals
of the DXR-substrate complex were grown using DXR at a
concentration of 10 mg/ml containing 3 mM NADPH
and 10 mM substrate. The reservoir buffer contained 25%
(w/v) PEG 3350, 200 mM Li2SO4, 100 mM Bis-Tris (pH
6.5). Crystals appeared overnight as thin plates of
maximum dimensions 0.1 mm!0.1 mm!0.03 mm and
were mounted directly from the microbatch plate.
For DXR-fosmidomycin complex crystals, wild-type
(non-SeMet-labelled) and SeMet-labelled E. coli DXR,
crystals were optimized using the hanging-drop vapor-
diffusion method at a protein concentration of 10 mg/ml,
containing 3 mM NADPH and 10 mM fosmidomycin.
The protein solution contained 100 mM NaCl, 50 mM
Tris–HCl (pH 8.0), 5% glycerol, 5 mM TCEP and 10 mM
MgCl2. The reservoir buffer contained 2.6 M NaCl,
100 mM acetate (pH 5.0). Crystals grew to a maximum
size of 0.1 mm!0.1 mm!0.1 mm over three days. 1.6 M
sodium citrate was used as a cryoprotectant for low
temperature measurements (with a soak time of 1–2
seconds).
Data collection and processing
The substrate complex crystals belong to the orthor-
hombic space group C2221 with cell dimensions aZ
107.57, bZ122.85, cZ128.55 Å. They contain two mol-
ecules in the asymmetric unit and diffract to a resolution
of 1.9 Å. The data set was collected at the SLS beamline
X06SA at PSI, Villigen, Switzerland and diffraction
intensities were recorded on a MAR CCD detector at a
wavelength of 0.97818 Å.
The fosmidomycin complex crystals belong to the
trigonal space group P3121 with cell dimensions aZbZ
Crystal Structure of DXR 125

Table 2. Data collection and refinement statistics

SeMet, NADPH, Wild-type, NADPH, Wild-type, NADPH,

fosmidomycin fosmidomycin substrate

Data collection
Space group P3(1)21 P3(1)21 C222(1)
Cell dimensions aZbZ109.17, cZ90.63 aZbZ109.14, cZ91.29 aZ107.06, bZcZ128.55
Limiting resolution (Å) (last shell) 2.20 (2.34–2.20) 2.65 (2.82–2.65) 1.90 (2.02–1.90)
Reflections 31,221 18,451 62,616
I/sI (overall/last shell) 7.64 (1.97) 12.1 (2.98) 12.6 (2.73)
Rmerge (%) (overall/last shell) 12.9 (46.5) 14.3 (44.4) 9.6 (35.9)
Redundancy 3.8 6.8 4.8
Completeness (%) (overall/last shell) 97.5 (94.5) 99.2 (95.5) 93 (95.3)
Refinement
Resolution 20–2.2 20–2.65 20–1.90
Rcryst (%) 18.7 21.1 17.4
Rfree (%) 21.2 25.8 21.2
Non-hydrogen protein atoms 3030 3030 6060
Solvent molecules 265 111 639
B-factor (Å2)
Protein 23.8 25.5 19.8
Solvent 32.0 25.4 32.0
Fosmidomycin 24.9 25.4 18.5
NADPH 49.5 33.5 14.6
Rmsd
Bond length (Å) 0.010 0.007 0.007
Bond angle (8) 1.175 1.063 1.136

109.17, cZ90.63 Å. They contain one molecule in the
asymmetric unit and diffract to a resolution of 2.65 Å for
the wild-type enzyme and 2.2 Å for the SeMet-labelled
protein. Data collection and refinement statistics are
summarized in Table 2. Both data sets were measured
with Cu Ka radiation provided from a NONIUS FR591
rotating anode generator equipped with an OSMIC
mirror system. Diffraction intensities were recorded on
a MAR-Research image plate area detector. All diffraction
data were further processed and scaled with DENZO and
SCALEPACK.32
structures with excellent stereochemistry. All residues
fall within the allowed regions of the Ramachandran plot
and meet all stereochemical requirements of the program
PROCHECK.38 The refined structure of the fosmido-
mycin-complexed E. coli enzyme has an R-factor of 18.7%
(RfreeZ20.2%) for the SeMet-labelled protein and 21.1%
(RfreeZ25.8%) for wild-type DXR (Table 2). The refined
structure of the substrate-complexed enzyme has an R-
factor of 17.4% (RfreeZ21.2%).
Protein Data Bank accession numbers

Molecular replacement and refinement
The ternary complex of E. coli DXR with NADPH and
fosmidomycin was solved by molecular replacement,
where phases were obtained with the AMoRe software33,34,
using the refined coordinates of the apoenzyme (PDB
accession code 1K5H, molecule A).15 The Eulerian angles
and translation parameters are aZ22.538, bZ38.298, gZ
128.618, xZK31.12 Å, yZ73.09 Å, zZK46.83 Å for the
single molecule in the asymmetric unit. The ternary
complex of DXR with NADPH and DXP was solved using
the coordinates of the fosmidomycin complex described
here. The Eulerian angles and translation parameters are
aZ88.28, bZ0.08, gZ0.08, xZ52.91 Å, yZK32.86 Å, zZ
2.80 Å for molecule A and aZ151.198, bZ0.008, gZ0.008,
xZ52.82 Å, yZ32.34 Å, zZ34.95 Å for molecule B of the
asymmetric unit.
Maximum likelihood refinement was performed with
REFMAC5,35 using parameters for ideal stereochemistry
as described.36 Manual rebuilding was accomplished
using the graphics program MOLOC.37 The initial
difference Fourier map of the fosmidomycin complex
clearly revealed a residual electron density corresponding
to fosmidomycin and a partially bound molecule of
NADPH, while that of the substrate-complexed enzyme
revealed a residual electron density corresponding to the
partially racemized substrate and NADPH.
Progressive introduction of ligand and solvent mol-
ecules with good geometry led to the complexed
The structures have been deposited with the RCSB
Protein Data Bank under the accession codes 1Q0H, 1Q0L
and 1Q0Q.
Acknowledgements
We thank Allan D’Arcy for excellent assistance in
protein crystallization. We also thank the staff of the
Swiss Light Source, Paul Scherrer Institute, Villigen,
Switzerland for assistance with data collection.
R.P.M.F. has a PhD scholarship from CNPq (Brazil).
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Edited by R. Huber