We report the preparation of13C-labeled substrate isotopologs that were designed to opti-mize the detection of an exchange of putative cleavage products that might occur in the hypotheti
Trang 1of 2C-methyl-D-erythritol-4-phosphate synthase
Susan Lauw, Victoria Illarionova, Adelbert Bacher, Felix Rohdich and Wolfgang Eisenreich
Center for Integrated Protein Research, Lehrstuhl fu¨r Biochemie, Department Chemie, Technische Universita¨t Mu¨nchen, Garching, Germany
Terpenes are the largest group of natural products,
comprising more than 35 000 compounds [1] They are
all biosynthesized from two simple precursors,
isopen-tenyl diphosphate and dimethylallyl diphosphate
These universal precursors were initially believed to be
biosynthesized exclusively via the mevalonate pathway
[2–4], but more recent studies have shown the existence
of a second pathway via 1-deoxy-d-xylulose
5-phos-phate (1) and 2C-methyl-d-erythritol 4-phos5-phos-phate (3)
(Fig 1) [5–9] This pathway is now known to supply the precursors for the isoprenoids of apicomplexan protozoa and of many eubacteria, as well as for the majority of isoprenoids from plants [10–14]
2C-Methyl-d-erythritol-4-phosphate synthase (IspC), encoded by the ispC gene (also designated dxr), cata-lyzes the first committed step in the nonmevalonate pathway [15] and has been shown to be the molecular target of fosmidomycin [16,17], an antibiotic from
Keywords
deoxyxylulose; dimethylallyl diphosphate;
isopentenyl diphosphate; terpene
Correspondence
W Eisenreich, Center for Integrated Protein
Research, Lehrstuhl fu¨r Biochemie,
Department Chemie, Technische Universita¨t
Mu¨nchen, Lichtenbergstr 4, D-85747
Garching, Germany
Fax: +49 89 289 13363
Tel: +49 89 289 13336
E-mail: wolfgang.eisenreich@ch.tum.de
F Rohdich, Center for Integrated Protein
Research, Lehrstuhl fu¨r Biochemie,
Department Chemie, Technische Universita¨t
Mu¨nchen, Lichtenbergstr 4, D-85747
Garching, Germany
Fax: +49 89 289 13363
Tel: +49 89 289 13336
E-mail: felix.rohdich@ch.tum.de
(Received 11 March 2008, revised 8 June
2008, accepted 11 June 2008)
doi:10.1111/j.1742-4658.2008.06547.x
2C-Methyl-d-erythritol-4-phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalo-nate isoprenoid biosynthetic pathway The reaction involves the isomeriza-tion of 1-deoxy-d-xylulose 5-phosphate, giving a branched-chain aldose derivative that is subsequently reduced to 2C-methyl-d-erythritol 4-phos-phate The isomerization step has been proposed to proceed as an intramo-lecular rearrangement or a retroaldol–aldol sequence We report the preparation of13C-labeled substrate isotopologs that were designed to opti-mize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol–aldol reaction sequence In reaction mixtures containing large amounts of 2C-methyl-d-erythritol-4-phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidop-sis thaliana, and a mixture of [1-13C1]-2C-methyl-d-erythritol 4-phosphate and [3-13C1]2C-methyl-d-erythritol 4-phosphate, the reversible reaction could be followed over thousands of reaction cycles No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m per catalytic cycle Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorpo-rated into the enzyme product In contrast to other reports, IspC did not catalyze the isomerisation of 1-deoxy-d-xylulose 5-phosphate to give 1-deoxy-l-ribulose 5-phosphate under any conditions tested However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis Although a retroaldol–aldol mechanism cannot be ruled out conclusively, the data show that a retrol-dol–aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three geneti-cally distant organisms
Abbreviation
IspC, 2C-methyl- D -erythritol-4-phosphate synthase.
Trang 2Streptomyces lavendulae [18,19] The development of
that compound as an antibiotic drug was aborted in
the 1980s, but recent work has shown activity against
various Plasmodium spp., including Plasmodium
fal-ciparum, a major human pathogen [17,20–22] These
studies have validated IspC as a target for the
develop-ment of novel antimalarial agents, which are urgently
needed in light of the enormous death toll of malaria
[23] and the rapid dissemination of variants with
resis-tance against currently available drugs [24] Moreover,
IspC and the consecutive enzymes of the pathway are
believed to be potential targets for the chemotherapy
of infections by a variety of eubacterial pathogens,
most notably Mycobacterium tuberculosis [14,25–27]
The first step of the reaction catalyzed by IspC has
been shown to give the branched aldose derivative,
2C-methyl-d-erythrose 4-phosphate, which is
subse-quently reduced to 2C-methyl-d-erythritol 4-phosphate
[28] The reductive reaction step has been shown to
involve the transfer of a hydride ion from the pro-S
position at C-4 of NADPH to the RE position of C-1
of reaction intermediate 2 (Fig 1) [29,30] The
forma-tion of 2 from the linear deoxyketose-type substrate
has been shown to proceed by cleavage of the bond
between C-3 and C-4 and the generation of a novel bond between C-1 and C-3 of the substrate [31,32] A sigmatropic rearrangement and a retroaldol–aldol reac-tion sequence are both compatible with the presently available data (Fig 2) [28,31–34], whereas a hydride shift mechanism has been ruled out by isotope labeling studies [35] Recently, the formation of 1-deoxy-l-ribu-lose 5-phosphate, an epimer of 1, was reported in an IspC-catalyzed reaction without NADPH and Mg2+
or Mn2+, and this observation was interpreted as evidence for a retroaldol mechanism of the reaction catalyzed by IspC [36] Here, we report on extensive stable isotope experiments aimed at discrimination between a sigmatropic rearrangement and a retro-aldol–aldol mechanism Additional mechanistic infor-mation on the enzyme-catalyzed reaction could benefit the development of novel inhibitors for use as anti-infective drugs
Results
IspCs of Escherichia coli, M tuberculosis and Arabid-opsis thalianawere selected for parallel enzyme studies, after a phylogenetic analysis of 31 IspC amino acid
Fig 1 Reactions catalyzed by IspC: 1,
1-deoxy- D -xylulose 5-phosphate; 2,
2C-methyl- D -erythrose 4-phosphate; 3,
2C-methyl- D -erythritol 4-phosphate.
Fig 2 Hypothetical mechanism of the
enzymatic reaction catalyzed by IspC: 1,
1-deoxy- D -xylulose 5-phosphate; 2,
2C-methyl-D -erythrose 4-phosphate; 4, glycolaldehyde
phosphate; 5, enolate of hydroxyacetone.
Trang 3sequences from prokaryotic and eukaryotic species had
shown the genetic distance between these three enzyme
species to be relatively large (Fig 3) The degrees of
sequence identity of the enzyme from E coli with the
orthologous enzymes from M tuberculosis and A
tha-liana are 40% and 43%, respectively Notably, the
study organisms are located in three different branches
of the dendrogram
As opposed to a sigmatropic rearrangement, a
retro-aldol–aldol reaction sequence can involve the exchange
of fragments between different substrate molecules,
unless the reaction proceeds in strict containment in a
reaction cavity that does not permit the escape and
reutilization of reaction intermediates The
hypotheti-cal retroaldol cleavage of 2C-methyl-d-erythritol
4-phosphate (3), according to Fig 4, should give
glycolaldehyde phosphate (4) and hydroxyacetone (5)
as reaction intermediates [33] In order to measure the
frequency of any potential intermediate exchange, we
prepared two substrate isotopomers of 3 that were specifically designed to maximize the sensitivity for the diagnosis of fragment exchange by 13C-NMR spectroscopy Specifically, [1-13C1 ]2C-methyl-d-erythri-tol 4-phosphate and [3-13C1]2C-methyl-d-erythritol 4-phosphate (3a and 3b, respectively, Fig 4) were obtained from [3,4-13C2]glucose and [2,5-13C2]glucose, respectively, by the enzyme-assisted one-pot reaction strategy described previously [37] An enzyme-mediated recombination of fragments 4, 5a, 4a and 5 generated from a mixture of [1-13C1]2C-methyl-d-erythrose 4-phosphate and [3-13C1]2C-methyl-d-erythrose 4-phos-phate (2a and 2b, respectively) via the proposed retroaldol–aldol mechanism should result in the forma-tion of four isotopolog species of 1-deoxy-d-xylulose 5-phosphate (1a–1d, Fig 4) Notably, the enzyme-mediated recombination of [1-13C1]glycolaldehyde (4a) and the enolate of [1-13C1]hydroxyacetone (5a) could then give [3,4-13C2]1-deoxy-d-xylulose 5-phosphate
Fig 3 Phylogenetic tree of IspCs from vari-ous organisms The consensus cladogram was constructed by neighbor-joining analysis from an alignment of IspC amino acid sequences from six plant species, one cya-nobacterium (Synechocystis sp.), one protist (P falciparum), and 23 eubacteria represent-ing different families Gaps were removed from the alignment, and the total number of positions taken into account was 327 The numbers at the nodes are the statistical confidence estimates computed by the bootstrap procedure The bar represents 0.134 percent accepted mutation distance.
Trang 4C
D
A
p.p.m.
Fig 4 NMR analysis of IspC assays using [1- 13 C1]2C-methyl- D -erythritol 4-phosphate and [3- 13 C1]2C-methyl-D-erythritol 4-phosphate (3a and 3b, respectively) as initial substrates 1a, [3- 13 C1]1-deoxy- D -xylulose 5-phosphate; 1b, [4- 13 C1]1-deoxy- D -xylulose 5-phosphate; 1c, [3,4- 13 C2 ]1-deoxy- D -xylulose 5-phosphate; 2a, [1-13C 1 ]2C-methyl- D -erythrose 4-phosphate; 2b, [3-13C 1 ]2C-methyl- D -erythrose 4-phosphate, 4, protonated glycolaldehyde phosphate (unlabeled); 4a, protonated [1- 13 C1]glycolaldehyde phosphate; 5, enolate of hydroxyacetone (unlabeled); 5a, enolate
of [1- 13 C1]hydroxyacetone 13 C-NMR signals of an incubation mixture without enzyme (A), and with IspC from E coli (B), M tuberculosis (C) and A thaliana (D), respectively The asterisks denote signals due to impurities.
Trang 5(1c) This double-labeled species would be detected via
satellite lines in the13C-NMR spectrum due to13C13C
coupling
In order to maximize the diagnostic sensitivity, we
decided to conduct the experiments under steady-state
conditions where reactants 1 and 3 are present in
simi-lar amounts at thermodynamic equilibrium For that
purpose, reaction mixtures containing 5 mm 3a, 5 mm
3b, 215 mm NADP+ and 0.15–0.25 mm IspC from
E coli, M tuberculosis or A thaliana were incubated
at pH 8 and 37C for 24 h and were monitored by
13C-NMR spectroscopy The partial reduction of
NADP+by the enzyme rapidly resulted in steady-state
conditions where the steady-state concentrations of 1
and 3 were approximately equal (Fig 4)
Conse-quently, the forward and the reverse reaction rate
under equilibrium condition were also bound to be
approximately equal Notably, the IspC enzymes were
present in very high (near-stoichiometric)
concentra-tions Under these conditions, the substrate molecules
should be engaged by enzyme molecules on a
near-permanent basis
The residual enzyme activity after 24 h of incubation
was measured after massive dilution of an aliquot of
the reaction mixture, using 1 as substrate The decrease
in activity during the 24 h incubation period was in
the range 27–37% for the three different enzymes
under study
From the starting conditions and the enzyme
stabil-ity measurements under our reaction conditions, it
follows that an average substrate molecule should
have passed through approximately 8800, 12 100 and
2400 forward–reverse cycles in the experiments with
enzymes from E coli, M tuberculosis and A thaliana,
respectively (supplementary Table S4)
For the equilibrium constant of the reaction
cata-lyzed by IspC as defined by Eqn (1), we obtained a
value of (2.8 ± 0.2)· 10)10m at pH 8.0 and 37C
This is well in line with a value of (4.6 ±
0.5)· 10)10m at pH 7.7 and 37C that had been
reported earlier [28]
K ¼½NADPHeq ½1eq ½H
þ
Figure 4 shows 13C-NMR signals of the reaction
mixtures prior to the addition of enzyme (Fig 4A) and
after incubation with enzymes from E coli (Fig 4B),
M tuberculosis (Fig 4C) and A thaliana (Fig 4D),
respectively Reaction mixtures treated with enzymes
from the three different organisms studied showed very
similar results
The crucial observation is the absence of any detect-able excess of the13C13C coupling satellites beyond the natural abundance level for the signals of C-3 and C-4
of a hypothetical product 1c The hypothetical posi-tions of the 13C13C coupling satellites expected in the spectrum of 1c are marked by arrows in Fig 4B–D In each case, the integrals of the satellite signals are in the range of 1% as compared to the central signal Signals of that size would be expected in the complete absence of fragment exchange, where they reflect the presence of about 1.1% 13C in those carbon atoms of the reactant that were not labeled
On the basis of the quantitative evaluation of the
13C-NMR signal intensities and coupling satellites in experiments with 13C-labeled substrates, it can be esti-mated that fewer than one fragment exchange has occurred during more than 100 000 reaction cycles Although these data are not sufficient to rule out a ret-roaldol–aldol reaction sequence, they do show that a hypothetical retroaldol–aldol sequence would require extremely tight confinement of the intermediary molec-ular fragments at the active site of the enzyme The limit for escape and reutilization of a retroaldol frag-ment would be fewer than once in 100 000 forward– reverse cycles In this context, it is also worth noting that the branched intermediate 2C-methyl-d-erythrose 4-phosphate (2) (Fig 1) can be used as substrate by the enzyme at a rate that is comparable with the con-version rate of substrate 1 [28]; thus, strict confinement seems at least not to apply to that intermediate
In a second set of experiments, we checked whether exogenous hydroxyacetone, whose enolate is the pre-dicted intermediate of the hypothetical retroaldol–aldol mechanism, can be incorporated into reactants 1 and 3
by fragment exchange Preliminary experiments had shown that hydroxyacetone does not significantly change the catalytic rate of 2C-methyl-d-erythritol 4-phosphate synthase when present in concentrations
up to 2% (v⁄ v) The reaction mixtures contained
10 mm [1,3,4-13C3]2C-methyl-d-erythritol 4-phosphate (3c, Fig 5), 215 mm NADP+, 243 mm (2%, v⁄ v) hydroxyacetone, 100 mm Tris⁄ HCl (pH 8.0), and 0.23–0.25 mm IspC from E coli, M tuberculosis or
A thaliana They were incubated for 8 h at 37C and were then analyzed by NMR spectroscopy
As described above, these initial conditions were rapidly conducive to steady conditions where 1 and 3 were present in very similar concentrations, and the rates of the forward reaction (conversion of 1 to 3) and the backward reaction (conversion of 3 into 1) were also essentially the same Any ‘wash-in’ of unlabeled hydroxyacetone (6) should give the iso-topolog 1f, which carries only two 13C atoms This
Trang 6B
C
D
p.p.m.
Fig 5 NMR analysis of IspC assays using [1,3,4-13C 3 ]2C-methyl- D -erythritol 4-phosphate and unlabeled hydroxyacetone (3c and 6) as initial substrates 1e, [3,4,5- 13 C3]1-deoxy- D -xylulose 5-phosphate; 1f, [4,5- 13 C2]1-deoxy- D -xylulose 5-phosphate; 3d, [3,4- 13 C2]2C-methyl- D -erythritol 4-phosphate; 4b, protonated [1,2- 13 C2]glycolaldehyde phosphate; 5, enolate of hydroxyacetone (unlabeled); 5a, enolate of [1- 13 C1 ]hydroxyace-tone; 6, hydroxyacetone (unlabeled) 13 C-NMR signals of an incubation mixture without enzyme (A), and with IspC from E coli (B), M tuber-culosis (C) and A thaliana (D), respectively The asterisks denote signals due to impurities.
Trang 7isotopolog would be diagnosed easily in the13C-NMR
spectra by a distinctive double doublet signature of
C-4 that would be caused by 13C13C coupling and
13C31P coupling (predicted signal positions are
indi-cated by arrows in Fig 5B–D)
Figure 5A–D shows13C-NMR signals detected in the
exchange experiment with unlabeled hydroxyacetone
Signal intensities showed a steady-state ratio of 59 : 41
for 1 and 3c (supplementary Table S6) The crucial
double doublets as expected for a retroaldol–aldol
mechanism (Fig 5B–D) were absent The 13C NMR
data of 1e and 3c are shown in supplementary Table S5
In the following set of experiments, we investigated
whether glycolaldehyde phosphate and hydroxyacetone
can serve as direct substrates for IspC from different
organisms to form 2C-methyl-d-erythritol 4-phosphate,
as shown in the hypothetical retroaldol–aldol reaction
sequence illustrated in Fig 5 Specifically, the reaction
mixtures contained 2 mm [1,2-13C2]glycolaldehyde
phosphate (4b), 243 mm (2%, v⁄ v) hydroxyacetone (5),
Tris⁄ HCl (pH 8.0), 3 mm NADPH, and 0.21–0.34 mm
IspC from E coli, M tuberculosis or A thaliana
The 13C-NMR spectra obtained after incubation periods of 1.5 and 3 h, respectively, showed only dou-ble doudou-blet signals at 89.2 p.p.m due to the presence
of the hydrate of 4b (Fig 6B–D) As shown in Fig 6,
no evidence for the formation of [3,4-13C2
]2C-methyl-d-erythritol 4-phosphate (3d) could be obtained Nota-bly, it would have been possible to detect any 3d by the specific double doublet signature of C-3, as confirmed
by a titration experiment with [1,3,4-13C3 ]2C-methyl-d-erythritol 4-phosphate (3c) (Fig 6E) It should be noted that these experiments were conducted with very high concentrations of enzymes (almost in the millimo-lar range) and with a very high concentration of
243 mm (2%, v⁄ v) of hydroxyacetone, which had been shown to be tolerated by the enzyme without significant reduction in the rate for the IspC reaction measured with 1e as substrate (see also supplementary Table S7) Wong & Cox [36] reported the formation of 1-deoxy-l-ribulose 5-phosphate (7b, Fig 7), an epimer
of 1, in an IspC reaction mixture in the absence of NADPH and of divalent metal ions Specifically, they observed a new 13C-NMR signal at 71.6 p.p.m., which
A
B
C
D
E
p.p.m.
Fig 6 NMR analysis of IspC assays using protonated [1,2- 13 C2]glycolaldehyde phos-phate and the enolate of hydroxacetone (4b and 5a, respectively) as initial substrates 2c, [3,4- 13 C2]2C-methyl- D -erythrose 4-phos-phate; 3d, [3,4- 13 C 2 ]2C-methyl- D -erythritol 4-phosphate (A)–(E) are13C-NMR spectra obtained from IspC reactions using [1,2- 13 C2]glycolaldehyde phosphate (4b) and hydroxacetone (5a) as substrates.13C-NMR signals of an incubation mixture without enzyme (A), and with IspC from E coli (B),
M tuberculosis (C) and A thaliana (D) and with the addition of [1,3,4- 13 C3]2C-methyl- D -erythritol 4-phosphate after 3 h of incubation
of the reaction mixture B (E) The asterisks denote signals due to impurities.
Trang 8was assigned to C-4 of 7 When we repeated that
experiment with [3,4,5-13C3]-1 as substrate, we found
that a signal was already present at 71.6 p.p.m., even
prior to incubation of the reaction mixture (Fig 8,
lane A), and the intensity of that signal increased by a
factor of about 2 during the subsequent incubation
Notably, the same phenomenon was observed in
sam-ples without IspC This unexpected finding prompted a
more detailed investigation, which revealed that 1 is
subject to spontaneous isomerization to give 7 The
details are described below
Specifically, we prepared [3,4,5-13C3]-1 by an
enzy-matic procedure starting from [U-13C6]glucose [37]
Despite the absence of IspC, the formation of the
target compound 1 was accompanied by the formation
of a compound characterized by a 13C resonance at
71.6 p.p.m., albeit at a much lower rate Specifically,
after incubation for 1 h, the yield of 1 was about 50%,
and the relative yield, based on 1, of the compound
resonating at 71.6 p.p.m was 1% Even after the
removal of all proteins by ultrafiltration, the relative
amount of that contaminant continued to increase over
a period of about 1 week; the final ratio of the two
compounds, believed to represent a state of
equilib-rium, was about 4 : 1 The apparent rate constant for
the formation of 7 from 1 was 3· 10)7s)1, and, the
equilibrium constant was calculated to be 3.45 (supple-mentary Fig S1) In parallel experiments with and without addition of IspC, the 13C signal at 71.6 p.p.m increased at the same rate More specifically, reaction mixtures containing 100 mm Tris⁄ HCl (pH 8), 10 mm
1, and 0.1 mm (5 mgÆmL)1) IspC when required, were incubated at 37C The component resonating at 71.6 p.p.m increased from 3% to 5% (based on the concentration of 1) during a period of 24 h at 37C, irrespective of the presence or absence of IspC
Using an equilibrium mixture of [U-13C5]-1 and of the component resonating at 71.6 p.p.m., we could assign all 13C signals of the latter on the basis of
13C13C and 13C31P coupling in one-dimensional 13 C-NMR spectra (supplementary Fig S2) All 1H-NMR signals of the newly formed compound were then assigned by HMQC spectroscopy (supplementary Table S8) The NMR data were in close correspon-dence with those reported earlier for a chemically synthesized sample of 1-deoxy-l-ribulose 5-phosphate (7b, Fig 7) [38] However, it should be noted that
13C-NMR is unable to discriminate between the
d-enantiomer and l-enantiomer under the experimental conditions used, and enantiomer assignment of the 1-deoxyribulose 5-phosphate formed by spontaneous isomerization of 1-deoxy-d-xylulose 5-phosphate (1) is not possible from our experimental data
Discussion
The main part of the present study was a search, under conditions of maximal stringency, for fragment exchange that could be the hallmark of the hypotheti-cal retroaldol–aldol mechanism The essentials of that high-stringency strategy can be summarized as fol-lows: (a) the experiments shown in Figs 4 and 5 were conducted under steady-state conditions (at thermody-namic equilibrium), thus enabling each molecule to pass through thousands of forward–backward reaction cycles; (b) enzymes were used at near-stoichiometric concentrations, in order to engage substrate molecules
on a near-permanent basis; (c) multiple 13C labeling was used in order to optimize detection of the crucial molecular species that would have resulted from frag-ment exchange by the utilization of 13C13C coupling; (d) substrates used included not only the natural sub-strate of the reaction, 1-deoxy-d-xylulose 5-phosphate (1), but also the hypothetical fragments that would be expected to result from a retroaldol fragmentation, i.e hydroxyacetone and glycolaldehyde phosphate – one of these substrates (glycolaldehyde phosphate, 4b) was double-labeled with 13C, and the other was used
at an unusually high concentration (in the decimolar
Fig 7 Stereoisomers of 1-deoxy- D -xylulose 5-phosphate: 1,
1-deoxy- D -xylulose 5-phosphate; 7a, 1-deoxy- D -ribulose
5-phos-phate; 7b, 1-deoxy- L -ribulose 5-phosphate; 8, 1-deoxy- L -xylulose
5-phosphate.
Trang 9range) in order to maximize diagnostic sensitivity; (e)
three IspC orthologs from genetically distant sources
were used, with the expectation that all orthologs
would not necessarily confine fragments with the same
degree of stringency – as it is unlikely that strong
selective pressure specifically enforced very high
degrees of stringency, it would not appear implausible
that different taxa might have enzymes with different
stringencies
On the basis of these results, the limit on observed
fragment escape and fragment reutilization is fewer
than one in many thousands of forward–reverse
cycles
The active site of IspC is located close to the
sur-face A flexible loop at the active site (amino acids
206–216) [39–42] is able to fold into at least three
dif-ferent conformations Specifically, in the apoenzyme
structure, this loop is unordered, whereas the structure
with bound NADPH, and especially the complex with
bound NADPH as well as 1-deoxy-d-xylulose
5-phos-phate (1), showed this loop to be well ordered and to
be closing the active site region of the enzyme (Fig 9)
On the other hand, it has also been demonstrated that
the branched intermediate 2 can access the active site
cavity from the bulk solvent and can then serve as a
substrate, obviously without hindrance from the said
loop [28] The available structural data are not a
suffi-cient basis to support a claim of absolute confinement
of the active site Notably, both hypothetical fragments
resulting from retroaldol cleavage would be small by
comparison with the branched aldose intermediate 2;
as the active site is even accessible to 2, one would expect that the hypothetical intermediates 4b and 5, which are both small by comparison, should be able to exchange with the bulk solvent
3-Deoxy-1 and 4-deoxy-1 have been shown to act as weak inhibitors, but not as substrates of IspC [28,38] Had these investigations resulted in the demonstration
of any (even low) substrate activity for the 4-deoxy compound, that would have ruled out the retroaldol mechanism Clearly, however, the reverse argument would be a logical fallacy; the failure of the 4-deoxy compound to act as a substrate could be due to a wide variety of reasons, and does not determine the mechanism
The claimed conversion of 1 into the epimer 7 by IspC in the absence of pyridine nucleotides and diva-lent metal ions could have been construed as support for a retroaldol mechanism Unfortunately, our results suggest that the formation of 7 in those experiments was incorrectly ascribed to the catalytic action of IspC, and reflected, in reality, the spontaneous, uncatalyzed epimerization of 1
In summary, our data are all consistent with a sig-matropic rearrangement, albeit they do not constitute definite proof However, it appears safe to say that the present experiments extend the degree of stringency to the limits of experimental feasibility as ultimately defined by the long-term chemical stability of the proteins, substrates and coenzymes involved
p.p.m.
Fig 8 13 C-NMR spectra of 1-deoxy- D -xylu-lose 5-phosphate and its diastereomer Spectra were recorded in time intervals of
2 days at 37 C (A) Day 0 (B) Day 2 (C) Day 4 (D) Day 6 (E) Day 8 The signals of [3,4,5- 13 C 3 ]1-deoxy- D -xylulose 5-phosphate (1e) are shown in orange, and those of [3,4,5- 13 C3] 1-deoxy- L -ribulose 5-phosphate (7b) in green.
Trang 10Experimental procedures
Materials
[U-13C6]Glucose was purchased from Isotec, Miami
Town-ship, OH; [3,4-13C2]-glucose and [2,5-13C2]glucose were
from Omicron Inc., South Bend, OH [3,4,5-13C3
]1-deoxy-d-xylulose 5-phosphate, [U-13C5]1-deoxy-d-xylulose
5-phos-phate and [1,3,4,-13C3]2C-methyl-d-erythrose 4-phosphate
were synthesized as described previously [37,43,44]
Hexokinase from yeast, triosephosphate isomerase from
rabbit muscle, glutamate dehydrogenase from bovine liver
and glucose dehydrogenase from Thermoplasma
acido-philum were from Sigma 1-Deoxy-d-xylulose-5-phosphate
synthase from Bacillus subtilis and 2C-methyl-d-erythritol
4-phosphate synthase (IspC) from E coli were prepared
by published procedures [37,43,44] The preparation of
IspC from A thaliana, fructose-1,6-biphosphate aldolase,
phosphofructokinase, glucose-6-phosphate isomerase from
E coli, 6-phosphogluconate dehydrogenase and
glucose-6-phosphate dehydrogenase from B subtilis is described
elsewhere [44–47] The recombinant proteins used for
substrate synthesis and enzyme assays are listed in supple-mentary Table S3
Construction of a recombinant strain for hyperexpression of the M tuberculosis ispC gene The ispC gene of M tuberculosis (accession no
gb BX842581.1) was amplified by PCR using the oligonucle-otides ispCMycSacivo and ispCMycPstIhi as primers and chromosomal M tuberculosis DNA as template The amplifi-cate was digested with the restriction endonucleases SacI and PstI, and the resulting fragment was ligated into the expres-sion vector pQE30, which had been digested with the same restriction enzymes The ligation mixture was electroporated into E coli XL1-Blue [48] cells, giving the recombinant strains XL1-pQEispCMyco and M15-pQEispCMyco Bacterial strains, plasmids and oligonucleotides used in this study are listed in supplementary Tables S1 and S2
Sequence determination DNA sequencing was performed by the automated dide-oxynucleotide method N-terminal peptide sequences were obtained by pulsed-liquid mode
Expression of recombinant IspC from
M tuberculosis The recombinant E coli strain XL1-pQEispCMyco was grown in LB broth containing ampicillin (180 mgÆL)1) as appropriate Cultures were incubated at 37C with shaking
At an attenuance of 0.7 (600 nm), isopropylthiogalactoside was added to a final concentration of 0.5 mm, and the cultures were incubated overnight at 30C The cells were harvested by centrifugation at 5000 g for 30 min at 4C on
an SLA-3000 rotor (Sorvall, Du Pont, Newton, CT), washed with 0.9% (w⁄ v) sodium chloride, and stored at)20 C
Preparation of recombinant IspC from
M tuberculosis The frozen cell mass (40 g) of the recombinant E coli strain XL1-pQEispCMyco was thawed in 200 mL of
100 mm Tris⁄ HCl (pH 8.0), containing 0.5 m sodium chloride, 20 mm imidazole hydrochloride, and 10% (v⁄ v) glycerol The cells were disrupted using a French press, and the suspension was centrifuged at 15 000 g for 30 min
at 40C on an SS-34 rotor (Sorvall) The supernatant was applied to a column of Ni-chelating Sepharose FF (column volume, 34 mL) that had been equilibrated with 100 mm Tris⁄ HCl (pH 8.0) containing 0.5 m sodium chloride,
20 mm imidazole hydrochloride, and 10% (v⁄ v) glycerol (flow rate, 3 mLÆmin)1) The column was washed with
100 mm Tris⁄ HCl (pH 8.0) containing 0.5 m sodium
A
B
Fig 9 Crystal structures of monomeric IspC from E coli (A)
Apoenzyme (Protein Data Bank file 1K5H [41]) (B) Enzyme in
com-plex with NADPH (orange) and 1-deoxy- D -xylulose 5-phosphate
(blue) (Protein Data Bank file 1Q0Q [39]) The flexible loop (residues
206–216) in both structures is shown in magenta.