Mechanistic studies of an isph-catalyzed reaction: Implications for substrate binding and protonation in the biosynthesis of isoprenoids

Article abstract of DOI:10.1002/anie.201104124

Chosen handle: Mechanistic studies of the IspH-catalyzed reductive dehydroxylation of 4-hydroxy-3-methyl-2-(E)-1-diphosphate (HMBPP) to isopentenyl diphosphate and dimethylallyl diphosphate suggest that both the 4-OH group and the double bond of HMBPP may contribute to the formation of substrate-IspH complex. Labeling studies now show that the 4-hydroxy group of the substrate plays the dominant role in positioning the substrate in the enzyme active site.

Full text of DOI:10.1002/anie.201104124

Communications
DOI: 10.1002/anie.201104124
Enzyme Mechanisms
Mechanistic Studies of an IspH-Catalyzed Reaction: Implications for
Substrate Binding and Protonation in the Biosynthesis of Isoprenoids**
Wei-chen Chang, Youli Xiao, Hung-wen Liu,* and Pinghua Liu*
Isoprenoids are found widely in nature and have remarkably
diverse structures.[1] They are utilized by all living organisms
for a variety of biological functions, including serving as
structural components of cell membranes, key constituents of
electron-transport chains, and hormones to regulate various
physiological processes.[2] Many isoprenoids, produced as
secondary metabolites, function as defense agents for the
producers and have been a rich source for human medi-
cines.[2,3]
Successive condensation of isopentenyl diphosphate (IPP,
1, Scheme 1) and dimethylallyl diphosphate (DMAPP, 2) to
construct an isoprenyl backbone of desired length is a
common step in the biosynthesis of all isoprenoids.[1a,4] For
decades, it was believed that the mevalonic acid (MVA)
pathway is the sole source of IPP and DMAPP in all
organisms.[5] Only recently, a second pathway, the deoxyxy-
lulose phosphate (DXP) pathway (also known as the methyl
erythritol phosphate (MEP) pathway) was discovered,[1b,c,6] in
which both IPP and DMAPP are coproduced from 4-
hydroxy-3-methyl-2-butenyl diphosphate (HMBPP, 3) cata-
lyzed by the IspH enzyme (Scheme 1A).[7–11] Since IspH is not
present in humans, and isoprenoids are essential for the
survival of many pathogenic microorganisms, IspH has
become an attractive target for the development of new
antimicrobial drugs.[12]
The IspH-catalyzed conversion of 3 to 1 and 2 is an overall
two-electron reductive dehydroxylation reaction. Previous
biochemical, spectroscopic, and structural studies of IspH
revealed the presence of a [4Fe-4S] cluster having a unique
iron site to which the 4-hydroxy group of HMBPP (3) is
anchored (see 4 in Scheme 1C).[10b] This iron–sulfur cluster
plays an essential role in electron transfer during IspH
catalysis.[8,13] A mechanism resembling that of Birch reduction
has been proposed for the IspH-catalyzed reaction (Sche-
me 1B).[8a,b,9,13] However, in view of the close proximity of the
C2–C3 double bond of 3 to the unique iron site of the [4Fe-4S]
[*] W.-c. Chang,[+] Prof. Dr. H.-w. Liu
Division of Medicinal Chemistry, College of Pharmacy
Scheme 1. A) The IspH-catalyzed C4-dehydroxylation reaction, B) a
and Department of Chemistry and Biochemistry
University of Texas at Austin, Austin, TX 78712 (USA)
E-mail: h.w.liu@mail.utexas.edu
possible mechanism of IspH-catalyzed reaction, and C) two models
3ꢀ
of the one-electron-reduced IspH–HMBPP complex. PPi=P2O6
.
Dr. Y. Xiao,[+] Prof. Dr. P. Liu
Department of Chemistry, Boston University
Boston, MA 02215 (USA)
E-mail: pinghua@bu.edu
cluster (roughly 2.9–3.0 ꢀ) in the crystal structure of the
IspH–HMBPP complex (Figure 1) and the results of ENDOR
studies of an IspH E126A mutant,[10] an alternative mecha-
nism involving h2 coordination between the C2–C3 double
bond and the reduced [4Fe-4S]+ cluster (see 5 in Scheme 1C)
was also proposed.[8c] To further investigate the mechanism of
[+] These authors contributed equally to this work.
[**] Financial support for this work was provided in part by grants from
the National Science Foundation (CAREER, CHE-0748504 to P.L.),
the National Institutes of Health (GM 093903 to P.L.), and the
Welch Foundation (F-1511 to H.-w.L.).
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this intriguing reaction, we prepared a substrate analogue,
3-(hydroxymethyl)but-3-en-1-yl diphosphate (7, Scheme 2),
which is expected to bind to the [4Fe-4S]+ cluster in two
different orientations (see 19 and 20 in Scheme 3) depending
on whether formation of an metallacycle intermediate is part
of the catalysis. We report herein on the experimental details
and the mechanistic implications of these studies. The
evaluation of the competence of 7 as an IspH substrate and
analysis of the protonation of the allylic anion intermediate
(6) shed new light on the mode of action of IspH.
Scheme 3. A) The conversion of 3-(hydroxymethyl)but-3-en-1-yl diphos-
phate (7) to IPP (1) by IspH. B) Two possible binding modes of [5-13C]-
7 in the active site of IspH, and the anticipated respective outcomes of
the pronotation step.
of 7 to 1 by IspH were measured using the methyl viologen
assay.[8b] The analysis yielded a kcat of (484 ꢁ 6.5) minꢀ1 and a
Km for 7 of (694 ꢁ 79) mm. The kcat value is comparable to that
of (604 ꢁ 17) minꢀ1 determined for the native substrate
HMBPP (3) under similar conditions. However, the Km of 7
is nearly 35-fold higher than that of 3, resulting in a 44-fold
reduction of the catalytic efficiency (kcat/Km) relative to that
of HMBPP (3). Although 7 is a poor substrate, this result
nevertheless demonstrates that the substrate of IspH does not
necessarily have to have a double bond in the middle of its
carbon skeleton as in 3. This finding challenges the proposed
metallacycle model since the olefin moiety in 7 is further away
from the apical iron atom of the [4Fe-4S] cluster (see 19 in
Scheme 3B) if the binding mode is the same as that observed
in the recent IspH–HMBPP complex.[10b]
The fact that IPP is the sole product of the reaction of 7
and IspH is clearly different from the reductive dehydrox-
ylation of 3 by IspH in which both IPP (1) and DMAPP (2)
are produced in a ratio of approximately 5:1.[7d,9b] This ratio is
different from the roughly 1:3 distribution of IPP and
DMAPP at thermodynamic equilibrium.[8a] The production
of both IPP and DMAPP from HMBPP by IspH may be
explained by the specific binding mode of 3 in the active site
of IspH.[10b] As shown by the crystal structure in Figure 1,
HMBPP (3) binds to IspH in a bent conformation with its 4-
OH group coordinated to the apical iron of the [4Fe-4S]
cluster and its five-carbon backbone sandwiched between the
Scheme 2. Reagents and conditions: a) DHP (1.20 equiv), CH2Cl2, 08C,
2 h, 95%; b) OsO4 cat., NMO (1.50 equiv), acetone/potassium phos-
phate (KPi) buffer (100 mm, pH 7.40)/THF 2:2:1, RT, 3 h, 90%;
c) TBDPSCl (1.10 equiv), imidazole (2.00 equiv), DMAP cat., CH2Cl2,
RT, 12 h, 83%; d) DMSO (3.00 equiv), (COCl)2 (1.50 equiv), Et3N
(5.00 equiv), CH2Cl2, ꢀ788C to RT, 1 h, 85%; e) PPh3CH3I (2.00 equiv),
nBuLi (1.90 equiv), THF, 08C to RT, 2 h, 80%; f) TBAF (2.00 equiv),
THF, 90%; g) Ac2O (4.00 equiv), pyridine, RT, 14 h, 91%; h) AcOH/
H2O/THF 3:3:1, RT to 508C, 5 h, 80%; i) TsCl (2.00 equiv), pyridine,
08C, 12 h, 90%; j) [N(nBu)4]3P2O7H (1.30 equiv), MeCN, RT, 5 h, 40%;
k) NaOH (2.50 equiv), 08C to RT, 48 h, 60%. NMO=4-methylmorpho-
line N-oxide, TBDPS=tert-butyldiphenylsilyl, DMAP=4-dimethylami-
nopyridine, TBAF=tetrabutylammonium fluoride, Ts=4-toluenesul-
fonyl.
The synthesis of 7 followed the reaction sequence
delineated in Scheme 2 (see the Supporting Information for
details). The capability of IspH to process 7 as a substrate was
determined by monitoring the progress of the reaction with
1H NMR spectroscopy.[8b, 9b] The only turnover product found
in the incubation is IPP (1, Scheme 3A), which was isolated
1
and verified by H NMR spectroscopy and high-resolution
mass spectrometry. The kinetic parameters for the conversion
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Communications
via an h2-alkenyl intermediate, as proposed by the metallo-
cycle mechanism, coordination of the double bond of 7 to the
iron–sulfur cluster may be a prerequisite to substrate
orientation in the active site (Scheme 1C). Consequently,
[5-13C]-7 would bind to IspH in a conformation represented
by 20. Subsequent protonation of 22 at the carbon closer to
OB (now C5) should afford 1b as the product.
The labeled substrate [5-13C]-7 was synthesized according
to the reaction sequence shown in Scheme 2, except [13C]-
PPh3CH3I was used in the conversion of 12 to 13 (see the
Supporting Information for details). The [5-13C]-labeled
product was incubated with IspH, and the reaction was
quenched at appropriate time intervals (60% and 100%
conversion). After IspH was removed, the incubation mixture
was analyzed by 13C NMR spectroscopy. As shown in Fig-
ure 2A, [13C]-7 by itself gives one enriched 13C signal at
111.6 ppm. When the reaction was run to completion (Fig-
ure 2B), only one product was obtained. The sole signal that
Figure 1. The active site of IspH with the 4-OH group of HMBPP (3)
bound to the [4Fe-4S] cluster. The distances between OB to C4 and Oc
to C2 are roughly 3.4 and 3.5 ꢀ, respectively (pdb code: 3KE8).
[4Fe-4S] cluster and the C1-pyrophosphate group in the
enzyme active site. With such geometric constraints and the
lack of a nearby proton source, it was proposed that the
terminal phosphate group of HMBPP serves as the proton
donor in the final step (6!1 and 2, in Scheme 1B) of the
dehydroxylation reaction,[8a,9b,10b] where the negative charge
of the proposed allylic anion intermediate (6) is delocalized
through C2, C3, and C4. Because OC and OB (see Figure 1)
are within 3.4–3.5 ꢀ of the C2 and C4 positions of HMBPP,
they are likely involved in the protonation at C2 and C4 to
yield IPP and DMAPP, respectively. This hypothesis is
consistent with the pro-S stereochemistry observed for the
C2-protonation step (to form IPP from HMBPP).[14] Unlike
OC, OB forms a hydrogen bond with a water molecule, which
is also within hydrogen-bonding distance to E126. Thus, the
ratio of IPP and DMAPP may simply reflect the different
protonation state of OB and OC in the enzyme–substrate
complex. Although the water molecule generated in the
dehydroxylation step may serve as an alternative proton
source at C4, the fact that incubation with HMBPP and its
monofluoro analogue afforded IPP and DMAPP in the same
ratio (approximately 5:1)[9b] is most consistent with the
pyrophosphate (or the water molecule between OB and
E126) serving as the proton source (see 21/22).
When compound 7 is used as the substrate, the negative
charge of the proposed allylic anion intermediate will be
delocalized among C3, C4, and C5 (21/22 in Scheme 3B)
instead of C2, C3, and C4 (6), as seen in HMBPP (3). Hence,
owing to the proximal location of OB to C3, C4, and C5, OB is
most likely the proton donor and protonation at either C4 or
C5 will yield IPP (1) as the sole product, consistent with the
experimental observations. However, since OB is located
closer to C4 (3.4 ꢀ) than to C5 (4.6 ꢀ), protonation is
expected to occur largely at C4. Taking advantage of the
anticipated preferential protonation at the site closer to OB,
we probed this process using [5-13C]-7. We anticipated that if
coordination of the 4-OH group of 7 to the [4Fe-4S] cluster is
the anchor that positions the substrate in the enzyme active
site (shown as 19 in Scheme 3B), protonation at C4 of the
allylic anion intermediate (21) would yield 1a when labeled 7
is used as the substrate. In contrast, if the reaction proceeds
Figure 2. 13C NMR analysis of the incubation of [5-13C]-labeled 7
(5.0 mm) with IspH in 100 mm NaPi, pH 8.0 at 378C: A) in the
absence of enzyme; B) reaction was run with 5.0 mm IspH to comple-
tion (quenched after incubation for 1 h); C) reaction was run to 60%
completion with 1.0 mm IspH (quenched after incubation for 30 min).
appears at 111.4 ppm can be assigned to the resonance of the
terminal methylene carbon of the [13C]-labeled IPP product
(1a). When the reaction was quenched at 60% conversion
(Figure 2C), signals for both labeled 7 and 1a were present.
Interestingly, there were no signals corresponding to [13C]-1b,
which should have an enriched signal in the region of
approximately 25 ppm (i.e., the chemical shift for the IPP
methyl group). These results are consistent with the proposal
that coordination of the 4-OH group to the apical iron site is
important in positioning the substrate for reaction with the
[4Fe-4S] cluster, and C4 of 7 is the protonation site in the
IspH-catalyzed dehydroxylation of 7 to give 1a.
These results are significant for two reasons. First, the
outcome of the protonation experiments with [5-13C]-7 (i.e.,
only 1a is produced from 7) provide evidence supporting a
catalytic role for the terminal phosphate group of the
substrate in the final protonation step of the IspH reaction.
Second, our data also shed new light on the interaction
between the substrate double bond and the [4Fe-4S] cluster,
which has been proposed to play an important role in IspH
catalysis.[8c] However, the precise nature of this interaction
has been controversial: it may be a transannular effect
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comparing the incubation results with 3 and 7, it is now clear
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Received: June 15, 2011
Published online: October 24, 2011
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Keywords: deoxyxylulose phosphate pathway ·
enzyme catalysis · isoprenoid biosynthesis · IspH enzyme ·
reaction mechanisms
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