Methylerythritol cyclodiphosphate (MEcPP) in deoxyxylulose phosphate pathway: Synthesis from an epoxide and mechanisms

Article abstract of DOI:10.1039/c0cc02594a

In this communication, we reported another unique IspG-catalyzed transformation, the production of its substrate, MEcPP, from (2R,3R)-4-hydroxy-3-methyl-2,3-epoxybutanyl diphosphate (Epoxy-HMBPP) when reductants are excluded from the reaction mixture.

Full text of DOI:10.1039/c0cc02594a

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COMMUNICATION
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Methylerythritol cyclodiphosphate (MEcPP) in deoxyxylulose phosphate
pathway: synthesis from an epoxide and mechanismsw
Youli Xiao,a Rodney L. Nyland II,b Caren L. Freel Meyersb and Pinghua Liu*a
Received 15th July 2010, Accepted 13th August 2010
DOI: 10.1039/c0cc02594a
In this communication, we reported another unique IspG-
catalyzed transformation, the production of its substrate,
MEcPP, from (2R,3R)-4-hydroxy-3-methyl-2,3-epoxybutanyl
diphosphate (Epoxy-HMBPP) when reductants are excluded
from the reaction mixture.
To date, several lines of evidence are consistent with the
plausibility of the epoxide reductive deoxygenation model.
First, it is known that synthetic [4Fe–4S] clusters catalyze
reductive deoxygenation of epoxides to alkenes.19 Second,
our initial characterization of (2R,3R)-4-hydroxy-3-methyl-
2,3-epoxybutanyl diphosphate (Epoxy-HMBPP, 2) revealed
that IspG catalyzes reductive deoxygenation of Epoxy-HMBPP
to HMBPP with an efficiency comparable to that of
MEcPP.20 Following our initial Epoxy-HMBPP studies,
Oldfield and co-workers further suggested, on the basis of
EPR studies, that a common organo-metallic transient species
en route to HMBPP was generated from both MEcPP and
Epoxy-HMBPP.21
Isoprenoids, one of the largest class of natural products, are
biosynthesized either through the classical mevalonate
pathway (MVA pathway in animals, yeast, or archaea) or
the recently identified deoxyxylulose phosphate pathway
(DXP pathway in green algae, eubacteria, and plants).1–4
The penultimate step of the DXP pathway is an IspG-catalyzed
reductive deoxygenation of methyl-D-erythritol-2,4-cyclo-
diphosphate (MEcPP, 1, Scheme 1) to (E)-4-hydroxy-3-
methylbut-2-enyl diphosphate (HMBPP, 3). MEcPP was
isolated almost a decade before its corresponding biosynthetic
enzyme was identified and it accumulates to high concentrations
(up to 50 mM) when bacteria are under oxidative stress
conditions.5–7 Recently, the enzyme (GcpE or IspG) that
utilizes MEcPP as the substrate was identified.8–11 Preliminary
biochemical studies indicate that IspG is a unique iron site-
containing [4Fe–4S] protein, and it catalyzes the reductive
deoxygenation of MEcPP to HMBPP (Scheme 1).11–14
The first step of the epoxide model (Scheme 1) is a nucleo-
philic attack by the C3 hydroxyl group at the C2 position,
which leads to the ring opening of MEcPP to Epoxy-HMBPP.
Reductive ring opening of Epoxy-HMBPP by the reduced
iron–sulfur cluster ([4Fe–4S])1+ generates a substrate-based
radical. Further reduction to a substrate-based carbanion
followed by water elimination affords HMBPP. In our
previous studies, we demonstrated that IspG indeed catalyzes
the reductive deoxygenation of Epoxy-HMBPP to HMBPP.
In addition, the kinetic parameters of IspG catalyzed reductive
deoxygenations of both MEcPP and Epoxy-HMBPP are in
close agreement.20 The similar kcat values (B20 minÀ1) for the
IspG-catalyzed reductive deoxygenation of both MEcPP and
Epoxy-HMBPP suggest that a step after Epoxy-HMBPP is the
rate-limiting step if Epoxy-HMBPP is indeed an intermediate
in the reductive deoxygenation of MEcPP (Scheme 1).20
Our previoulsy reported steady state kinetic analysis of the
epoxide20 and Oldfield’s recent report suggesting MEcPP
and Epoxy-HMBPP proceed via a common downstream
intermediate21 are consistent with the epoxide model.
However, there is no other available information related to
the Epoxy-HMBPP intermediacy in the IspG-catalyzed
reductive deoxygenation of MEcPP, including the direct
detection of the epoxide in this reaction. The overall reactions
of MEcPP or Epoxy-HMBPP to HMBPP are reductive
deoxygenation processes and two electrons are supplied by a
reduction system. Under steady-state conditions and using
dithionite and methyl viologen as the reduction system, we
did not detect any accumulation of Epoxy-HMBPP during the
reductive deoxygenation of MEcPP to HMBPP.13,20 However,
according to the epoxide model (Scheme 1), the first step,
MEcPP to Epoxy-HMBPP conversion, does not involve any
redox chemistry. We hypothesized that if Epoxy-HMBPP
is indeed an intermediate along the MEcPP reductive
deoxygenation pathway, there might be a better chance of
detecting an IspG-catalyzed inter-conversion between MEcPP
and Epoxy-HMBPP by blocking the subsequent steps, which
Several mechanistic models were proposed to account for
the reductive deoxygenation of MEcPP to HMBPP, including
the vitamin K epoxyquinone reductase model,12 the anaerobic
ribonucleotide reductase (RNR) model,12,16 the deoxygenation
model utilized in ascarylose biosynthesis,17 the cation model,14,18
and the epoxide model proposed by Rohdich et al. (Scheme 1).15
Scheme 1 The epoxide model proposed by Rohdich et al. for
IspG-catalyzed reductive deoxygenation of MEcPP to HMBPP.15
a Department of Chemistry, Boston University, MA 02215, USA.
E-mail: pinghua@bu.edu; Fax: +1 617-353-6466;
Tel: +1 617-353-2481
b Department of Pharmacology and Molecular Sciences,
Johns Hopkins University School of Medicine, Baltimore,
MD 21205, USA
w Electronic supplementary information (ESI) available: Details of
experimental procedure. See DOI: 10.1039/c0cc02594a
c
7220 Chem. Commun., 2010, 46, 7220–7222
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Scheme 2 Two potential scenarios to account for IspG-catalyzed
reductive deoxygenations of both MEcPP and Epoxy-HMBPP to
HMBPP. (A) Epoxy-HMBPP as an intermediate in the reductive
deoxygenation of MEcPP to HMBPP; (B) MEcPP as an intermediate
in the reductive deoxygenation of Epoxy-HMBPP to HMBPP.
Fig. 2 IspG-catalyzed production of MEcPP from Epoxy-HMBPP.
1H-NMR spectra of the C5 methyl group region. Spectrum A:
Epoxy-HMBPP C5 methyl group region (in 100 mM Tris, pH 8.0);
Spectrum B: IspG catalyzed MEcPP production from Epoxy-HMBPP
(5.0 mM of [4Fe–4S]2+-containing IspG, 1.5 mM Epoxy-HMBPP in
100 mM Tris, pH 8.0 at 37 1C for 4 hours).
can be achieved by excluding the reduction system (e.g.,
NADPH–flavodoxin–flavodoxin reductase or dithionite/methyl
viologen). Thus, we examined two scenarios regarding the
relationship between MEcPP and Epoxy-HMBPP in IspG-
catalyzed reductive deoxygenation reactions (Scheme 2A
and B): First, production of Epoxy-HMBPP from MEcPP
(Scheme 2A), and second, MEcPP as an intermediate in
the reductive deoxygenation of Epoxy-HMBPP to HMBPP
(Scheme 2B).
1
was observed in the H-NMR spectrum (1.32 ppm, Fig. 2B),
downfield from the 1.25 ppm signal (C5 methyl group of
Epoxy-HMBPP, Fig. 2A). The new species has the same
chemical shift as that of the MEcPP methyl group. This new
species was isolated and its spectroscopic properties are
identical to that of authentic synthetic MEcPP (Fig. S1, ESIw).
We further characterized the diphosphate-containing product
using 1H–31P–31P COSY as previously reported.22 These
results are also consistent with the IspG-catalyzed production
of MEcPP from Epoxy-HMBPP (Scheme 2B and Fig. S2 in
ESIw). In the absence of IspG, there is no MEcPP production
from Epoxy-HMBPP, at least at our 1H-NMR assay
detection level.
The inter-conversion between MEcPP and Epoxy-HMBPP
reaction was monitored using our recently reported 1H-NMR
assay.13,20 The C5 methyl group chemical shifts in the
1H-NMR spectra for MEcPP (1.32 ppm) and Epoxy-HMBPP
(1.25 ppm) are well-resolved and can be used to monitor
Epoxy-HMBPP formation (Fig. 1 and 2). The as-isolated
[4Fe–4S]2+-containing IspG (5 mM) was incubated with
MEcPP (1.7 mM) at 37 1C, and the reaction was monitored
The kinetic parameters for the conversion of Epoxy-
1
HMBPP to MEcPP were measured using the H-NMR assay
with [4Fe–4S]2+-containing IspG as the catalyst (Fig. S3,
1
by H-NMR (Fig. 1). Over the course of 4 hours under these
conditions, we did not detect the formation of Epoxy-HMBPP
1
(Fig. 1B). For the H-NMR assay, Epoxy-HMBPP at 20 mM
ESIw). Under presumed saturating substrate conditions
(1.0 mM of Epoxy-HMBPP) and at various [4Fe–4S]2+
-
can be reliably detected. Our results from this study suggest
that Epoxy-HMBPP is either not produced from MEcPP
under these conditions or its production is at a level below
containing IspG concentrations (2.5 mM–10.0 mM IspG,
100 mM Tris–HCl, pH 8.0 at 37 1C), the kcat value for this
conversion (B2.0 minÀ1) was calculated using the average kcat
at various IspG concentrations (Fig. S4, ESIw). Under these
conditions, the kcat value (B2.0 minÀ1) is an order of magni-
tude lower than that of the reductive deoxygenation of Epoxy-
HMBPP to HMBPP (20.1 minÀ1).
1
our H-NMR assay detection limit.
Because Epoxy-HMBPP is an IspG substrate, we also
examined the reversibility of this reaction, MEcPP production
from Epoxy-HMBPP (Scheme 2B, Fig. 2). Interestingly,
when the [4Fe–4S]2+-containing IspG was incubated with
Epoxy-HMBPP in the absence of reductants, a new species
Previously, we demonstrated that IspG catalyzes a reductive
deoxygenation of both MEcPP and Epoxy-HMBPP to
HMBPP (Scheme 3).13,20 In this study, we have examined
two mechanistic options that might explain the IspG-catalyzed
reductive deoxygenation of both MEcPP and Epoxy-HMBPP
to HMBPP (Scheme 2): (1) Epoxy-HMBPP as an intermediate
in MEcPP reductive deoxygenation (Scheme 2A), and (2)
MEcPP as an intermediate in the reductive deoxygenation of
Epoxy-HMBPP (Scheme 2B). Our attempts to detect
the formation of Epoxy-HMBPP from MEcPP under both
reductive deoxygenation conditions and in the absence of
excess reductants were not successful. Subjecting MEcPP to
[4Fe–4S]2+-containing IspG did not result in the production
of Epoxy-HMBPP to a level detectable in our 1H-NMR assay.
Importantly, we detected an IspG-catalyzed conversion of
Epoxy-HMBPP to MEcPP in the absence of reductants
(Fig. 2 and Scheme 3). When chemical micro-reversibility is
Fig. 1 1H-NMR spectra of MEcPP (1) C5 methyl group region. The
reaction mixture contained 5.0 mM of [4Fe–4S]2+-containing IspG,
1.7 mM MEcPP (1) at 37 1C in 100 mM Tris, pH 8.0 in the absence
of extra reductants (e.g., dithionite/methyl viologen). (A) 5 min;
(B) 4 hours.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7220–7222 7221

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This work was supported in part by funding to P.L. from
NSF CAREER (CHE-0748504) and NIH (GM-093903).
C.F.M. was supported by the Johns Hopkins Malaria
Research Institute Pilot Grant. We thank Yan Sun for providing
technical support in the H, P-NMR characterization
of MEcPP.
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Scheme 3 IspG-catalyzed reactions. (A) IspG-catalyzed reductive
deoxygenation of MEcPP and Epoxy-HMBPP; (B) IspG-catalyzed
MEcPP formation.
taken into consideration, the production of MEcPP from
Epoxy-HMBPP implies that the inter-conversion to MEcPP
from Epoxy-HMBPP might be chemically feasible. However,
the steady-state kinetic analyses presented here are not
sufficient to support the intermediacy of either MEcPP or
Epoxy-HMBPP in IspG-catalyzed reductive deoxygenation
reactions. Pre-steady state analysis in the future will shed light
on the putative intermediacy of Epoxy-HMBPP in this
reaction.
In our studies, we did not detect Epoxy-HMBPP formation
from MEcPP. However, MEcPP is indeed produced from
Epoxy-HMBPP, albeit at a steady state rate that is an order
of magnitude lower than the kcats of reductive deoxygenation
of both MEcPP and Epoxy-HMBPP. One way to interpret
these results is that the slow steady state kcat does not support
an epoxide intermediate as suggested in the epoxide model
(Scheme 1). If that is the case, the reductive deoxygenation of
MEcPP and Epoxy-HMBPP might be achieved through
parallel pathways. Alternatively, it is possible that MEcPP is
a transient, and/or tight binding intermediate during catalysis;
it may not be released into solution at all or may dissociate
from IspG active site at a very slow rate. Similar scenarios
have been suggested in IspC catalysis, an early step of the
DXP pathway,23,24 and in acetohydroxy acid isomeroreductase
catalysis in the biosynthesis of branched-chain amino acids.25
The IspC-catalyzed reduction–isomerization reaction is
believed to proceed via a transient aldehyde intermediate.
However, the aldehyde intermediate has, to date, not been
directly observed.
While this manuscript was in preparation, following our
initial report on Epoxy-HMBPP studies,20 a report by Oldfield
and co-workers suggested that a common organo-metallic
transient species en route to HMBPP was generated from
both MEcPP and Epoxy-HMBPP based on EPR studies.21
Observation of the same downstream intermediate from
MEcPP and Epoxy-HMBPP could support a mechanism
where MEcPP proceeds through Epoxy-HMBPP en route to
HMBPP. However, as Epoxy-HMBPP has not been directly
observed, it is still possible that MEcPP and Epoxy-HMBPP
could produce a common downstream intermediate via
parallel pathways.
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