Mechanistic studies of IspH in the deoxyxylulose phosphate pathway: Heterolytic C-O bond cleavage at C4 position

Article abstract of DOI:10.1021/ja710245d

Isoprenoids are one of the largest and most structurally diverse groups of metabolites in nature. Their biosyntheses require two precursors, isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). There are two different pathways

Full text of DOI:10.1021/ja710245d

Published on Web 01/25/2008
Mechanistic Studies of IspH in the Deoxyxylulose Phosphate Pathway:
Heterolytic C-O Bond Cleavage at C4 Position
Youli Xiao,† Zongbao K. Zhao,‡ and Pinghua Liu*,†
Department of Chemistry, Boston UniVersity, Boston, Massachusetts 02215, and Dalian Institute of Chemical
Physics, CAS, Dalian 116023, P.R. China
Received November 16, 2007; E-mail: pinghua@bu.edu
Scheme 1. Two IspH Reaction Models
Isoprenoids are one of the largest and most structurally diverse
groups of metabolites in nature. Plants alone produce more than
30,000 isoprenoids.1 The biosynthesis of isoprenoids requires two
precursors, isopentenyl diphosphate (IPP, 2) and its isomer,
dimethylallyl diphosphate (DMAPP, 3, Scheme 1). There are two
different pathways to synthesize 2 and 3: the deoxyxylulose
phosphate (DXP) pathway and the mevalonic acid (MVA) path-
way.2 More importantly, these two pathways have a well-defined
distribution among different kingdoms. Most pathogenic bacteria
and protozoan parasites utilize the DXP pathway, while animals
synthesize their isoprenoid precursors from acetyl-CoA via the
MVA pathway. Plants have both DXP and MVA pathways.2 Thus,
mechanistic studies on the DXP pathway enzymes may lead to the
development of mechanism-based inhibitors as herbicides, broad-
spectrum antibiotics, and antimalaria drugs.3,4
Scheme 2. Evaluation of Two IspH Substrate Analogues
IspH in the DXP pathway catalyzes the reductive dehydration
of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP, 1) to
form 2 and 3. Studies in the past few years led to the establishment
of the in vitro Escherichia coli IspH catalytic system, which includes
IspH, flavodoxin, and flavodoxin reductase.5-12 The iron-sulfur
cluster containing E. coli IspH, a 36 kDa protein, has only three
conserved Cys residues (C12, C96, and C197).11 Replacement of
any of the three conserved cysteine residues reduced the catalytic
activity by a factor of more than 70,000.11 Recent EPR studies
suggest that the isolated IspH has a [3Fe-4S]+ cluster, which is
(5) (Scheme 2), with a C-F or C-P linkage, respectively, are used
to study the IspH-catalyzed C-O bond cleavage mechanism at both
C1 and C4 positions. Our results clearly demonstrated that C4 and
not C1 heterolytic C-O bond cleavage occurs and that the C4
hydroxyl group is involved in substrate binding.
attributed to the loss of the unique iron-site iron from a [4Fe-4S]
10-11
cluster.7,
In this work, we studied the IspH-catalyzed C-O
bond cleavage mechanism. The activities at both C1 and C4 positions
are examined.
We have overproduced IspH in E. coli and purified the protein
anaerobically to near homogeneity. The UV-vis spectrum of the
anaerobically purified IspH is consistent with the presence of iron-
sulfur clusters, e.g., the features at around 320 and 410 nm (Figure
1S). Under optimized conditions (Supporting Information), IspH
protein with an A410/A280 ratio of 0.4, close to that reported in
literature, can be reproducibly obtained without reconstitution.11
Purified IspH was characterized by two assays, the 1H NMR assay
to directly analyze the products, and the NADPH consumption assay
at 340 nm to determine the kinetic parameters using NADPH-
flavodoxin-flavodoxin reductase as a reducing system. The purified
IspH has a kcat of 11.6 min-1 and a Km lower than 15 µM, which
are close to the reported data.11 The ratio of 2 to 3 produced from
1 was determined to be 5:1 (Figure 2S), which is similar to the
reported 4.5:1 ratio.11 For enzymatic transformation of pyrophos-
phate-containing substrates, divalent metals such as Ca2+ or Mg2+
are normally required. We examined the effect of divalent metals
on IspH activity, and no significant effects were observed, which
was consistent with that reported.11
Recently, Rodich et al. proposed a mechanism (model A in
Scheme 1) to explain this unusual transformation.7 In this model,
it is believed that conformation restrictions at the enzyme active
site favor the C4 hydroxyl group as the leaving group instead of
the better leaving group, pyrophosphate at the C1 position.13 In
addition, the C4 hydroxyl group is directly ligated to the unique
iron site of the iron-sulfur cluster to facilitate this process. In this
model, the iron-sulfur cluster is involved in both the dehydration
and reduction steps. To generate the proposed allylic radical
intermediate, it goes through the reduction of the double bond to
produce a radical anion intermediate, which is then used to mediate
the dehydration reaction. The resulting allylic radical intermediate
is then reduced to the products (2 and 3). Model A (Scheme 1) is
not the only route to the reductive dehydration products, 2 and 3.
Another option (model B in Scheme 1) is through a deprotonation-
mediated dehydration reaction as found in aconitase, followed by
reductions.
Two mechanistic probes, (E)-3-(fluoromethyl)-2-butenyl diphos-
phate (4) and (E)-4-hydroxy-3-methyl-2-butenyl pyrophosphonate
Substrate analogue 4 (Scheme 2) with a fluorine atom replacing
the C4 hydroxyl group was synthesized according to a literature
procedure.14 Because of the strong electronegativity of the fluorine
† Boston University.
‡ Dalian Institute of Chemical Physics.
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10.1021/ja710245d CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
atom, the C-F bond cleaves heterolytically. If the formation of
the allylic radical in Scheme 1 is a one-step reductive homolytic
C-O bond cleavage reaction, 4 will only be an inhibitor, and no
enzymatic turnover is expected. Substrate analogue 5 (Scheme 2),
with a pyrophosphonate function group (C-P bond) at the C1
position, was also prepared (Scheme 1S). Because of the C-P bond
stability, 5 will only be an inhibitor, and no enzymatic turnover
will occur if C-O bond cleavage at the C1 position is one of the
steps in catalysis.
In conclusion, IspH does not fall into the two known classes of
unique iron-site-containing iron-sulfur proteins. Biochemical data
reported herein provide crucial evidence to not only support the
integrity of the C1 position C-O bond during reaction, but also
suggest a heterolytic C-O bond cleavage at the C4 position for
IspH-catalyzed reductive dehydration reaction. Also, the nearly 200-
fold increase in Km with 4 relative to 1 indicates that the C4 hydroxyl
group is involved in substrate binding. We are currently carrying
out more in-depth investigations to understand the unique IspH-
catalyzed transformations, the compositions of the iron-sulfur
cluster, and its role in catalysis.
Interestingly, both 4 and 5 are IspH substrates (Scheme 2), with
a kcat of 0.55 min-1 and a Km of 3.95 mM for 4 and a kcat of 0.44
min-1 and a Km lower than 15 µM for 5. IspH converts 4 to a
mixture of 2 and 3 in a ratio of 7:1, which is slightly higher than
the ratio of products produced from 1. The release of a fluoride
anion as the product from 4 was directly detected using 19F NMR
Acknowledgment. This work is supported by the Boston
University startup fund for P.L. We thank Professor Dennis Dean’s
laboratory at Virginia Polytechnic Institute for providing pDB1281
plasmid. We also thank Mr. Lingdong Li for his participation in
this work.
1
(Figure 3S). H NMR analysis of HPLC-purified product from 5
implies that only 6 is produced, and its identity was confirmed by
comparison with the synthetic standards (Figures 4S and 5S).
The results from the above studies are mechanistically informa-
tive and provide the reactivity information at both C1 and C4
positions. Recent EPR studies suggest that IspH is a unique iron-
site-containing [4Fe-4S] cluster protein.11 However, the IspH-
catalyzed reaction does not fall into any of the two well-studied
categories of these unique proteins. The first class is the dehydratase
family. The dehydration reactions catalyzed by this class of enzymes
do not involve redox chemistry. Substrates coordinate to the [4Fe-
4S]2+ cluster at the unique iron site, and the [4Fe-4S]2+ cluster
serves as a Lewis acid to facilitate the heterolytic C-O bond
cleavage.15,16 Many examples of this class of enzyme are known,
and one of the best characterized enzymes is aconitase.16 The other
class is the radical SAM superfamily members,17-22 where SAM
coordinates to the [4Fe-4S]+ cluster at the unique iron site. One-
electron transfer from [4Fe-4S]+ to SAM leads to the production
of an adenosyl radical, which is then used to mediate many
energetically challenging reactions.23-28
For 4, the Km is increased by at least 200-fold compared to that
of 1, while the kcat is only about 4.7% that of 1. The Km increase
and kcat decrease observed for 4 are all consistent with the known
chemistry at the [4Fe-4S]2+ cluster unique iron site, such as that
of aconitase.16 Therefore, the C4 hydroxyl group of 1 is most likely
a ligand of the [4Fe-4S]2+ cluster unique iron site as it is in
aconitase. Replacing the hydroxyl group by a fluorine atom, which
is a weaker ligand, disrupts the interaction and causes a dramatic
increase in Km. Also, because the iron-sulfur cluster in the
aconitase-type of enzymes serves as a Lewis acid to facilitate the
dehydration process, the lack of this driving force in 4 explains
the significantly lower activity (kcat). In addition, because the strong
electronegative nature of the fluorine atom precludes a homolytic
C-F bond cleavage in 4, the production of 2 and 3 from 4 suggests
that the C4 position C-O bond is heterolytically cleaved as
suggested in Scheme 1.
Supporting Information Available: Procedures of IspH purifica-
1
tion and assays; H NMR spectra of 1, 4, 5, 6; 19F NMR spectra of 4
and fluoride anion (NaF solution); synthetic scheme of 5. This material
is free of charge via the internet at http://pubs.acs.org.
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involved in the reaction as suggested in model B of Scheme 1. It
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