IspG converts an epoxide substrate analogue to (E)-4-hydroxy-3-methylbut-2- enyl diphosphate: Implications for IspG catalysis in isoprenoid biosynthesis

Article abstract of DOI:10.1021/ja907470n

(Chemical Equation Presented) IspG is an intriguing enzyme in bacteria, parasite, and plant isoprenoid biosynthesis, and its catalytic mechanism remains elusive. We report here the synthesis of (2R,3R)-4-hydroxy-3-methyl-2,3- epoxybutanyl diphosphate (Epoxy-HMBPP), a proposed intermediate in one of the frequently cited mechanistic models. We have also demonstrated that this epoxide analogue is a catalytically competent IspG substrate. This study represents the first mechanistic study of this important enzyme.

Full text of DOI:10.1021/ja907470n

Published on Web 11/17/2009
IspG Converts an Epoxide Substrate Analogue to
(E)-4-Hydroxy-3-methylbut-2-enyl Diphosphate: Implications for IspG
Catalysis in Isoprenoid Biosynthesis
Rodney L. Nyland II,† Youli Xiao,‡ Pinghua Liu,*,‡ and Caren L. Freel Meyers*,†
Department of Pharmacology and Molecular Sciences, Johns Hopkins UniVersity School of Medicine, Baltimore,
Maryland 21205, and Department of Chemistry, Boston UniVersity, Boston, Massachusetts 02215
Received September 9, 2009; E-mail: pinghua@bu.edu; cmeyers@jhmi.edu
Isoprenoids comprise a vast and structurally diverse class of
natural products that are assembled from two simple precursors,
isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate
(DMAPP). Essential for all living organisms, IPP and DMAPP are
biosynthesized by two biosynthetic pathways. Animals, fungi, and
archae bacteria utilize the mevalonate pathway for production of
IPP and DMAPP,1 whereas bacteria, parasites, and plants rely upon
the methylerythritol phosphate (MEP) pathway (Scheme 1) for
biogenesis of these two central precursors.2-8 Its essentiality and
prevalence in plants and human pathogens renders the MEP pathway
an attractive new target for the development of herbicides and anti-
infective agents.
flavodoxin reducing system.6 This dramatic improvement in IspG
activity permits more detailed studies to elucidate the IspG
catalytic mechanism.
Scheme 2. IspG Mechanistic Model Proposed by Rohdich et al.11
Scheme 1. Methylerythritol Phosphate (MEP) Pathwaya
Several mechanisms have been proposed for IspG.8,10-12
A
particularly intriguing and frequently cited mechanism was proposed
by Rohdich et al.11 and involves the C2-hydroxyl-assisted ring
opening of MEcPP (Scheme 2) to generate an epoxy intermediate,
(2R,3R)-4-hydroxy-3-methyl-2,3-epoxybutanyl diphosphate (Epoxy-
HMBPP, 10). The epoxide intermediate 10 is then converted to
HMBPP via a reductive dehydration process. While the deoxygen-
ation of epoxides to the corresponding olefins by a synthetic
[4Fe-4S]2+ cluster has been demonstrated13 and provides chemical
precedence for such a mechanism, there is no evidence to date that
IspG can convert the proposed expoxide intermediate 10 to HMBPP
(7). Here we report the synthesis and characterization of 10 and
demonstrate that it is catalytically competent as a substrate for IspG
in the formation of HMBPP.
a Abbreviations: GAP, glyceraldehyde phosphate; DXP, deoxyxylulose
phosphate; CDP-ME2P, 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phos-
phate; CDP-ME, methylerythritol cytidinyl diphosphate; MEcPP, methyleryth-
ritol cyclic diphosphate; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate.
Scheme 3. Synthesis of Epoxide Analogue 10a
Among the most mechanistically intriguing transformations
of the seven-step MEP pathway is the penultimate step catalyzed
by IspG (Scheme 1). IspG utilizes a cyclic diphosphate,
methylerythritol cyclic diphosphate (MEcPP, 6), as substrate for
a reductive ring-opening reaction to form (E)-4-hydroxy-3-
methylbut-2-enyl diphosphate (HMBPP, 7). Recently, we greatly
enhanced the IspG enzymatic activity relative to that reported
in previous work.9 With the use of dithionite as the reductant in
combination with a proper mediator to facilitate the reduction
of the IspG Fe-S cluster, the IspG activity was increased by
∼20-fold relative to that of the NADPH-flavodoxin reductase-
a Conditions: (a) Imidazole (2.5 equiv), TBDPSiCl (1.1 equiv), 5:1 DMF/
CH2Cl2, 0 °C to rt, 15 h, 100%. (b) SeO2 (0.5 equiv), t-BuOOH, CH2Cl2, 0 °C
to rt, 4 h. (c) NaBH4, MeOH, 0 °C, 5 min, 64% over two steps. (d) NaH,
BnBr, THF, 0 °C to rt, 15 h, 95%. (e) TBAF, THF, rt, 30 min, 86%. (f) (-)-
D-DET (2 equiv), Ti(OiPr)4 (1.5 equiv), t-BuOOH (3 equiv), CH2Cl2, -25 to -20
°C, 14 h, 63%. (g) MsCl, Et3N, CH2Cl2, -78 °C, 30 min, 98%. (h) Pd/C, 1:24
HCO2H/MeOH, rt, 1-12 h, 76%. (i) P2O7H·3NBu4, CH3CN, rt, 30 min, 40%.
† Johns Hopkins University School of Medicine.
‡ Boston University.
9
17734 J. AM. CHEM. SOC. 2009, 131, 17734–17735
10.1021/ja907470n  2009 American Chemical Society

C O M M U N I C A T I O N S
Compound 10 was synthesized as shown in Scheme 3. Benzyl
ether 15 was prepared from dimethylallyl alcohol in 52% yield over
five steps. Stereoselective incorporation of the epoxide moiety into
compound 16 was accomplished using Sharpless epoxidation
conditions.14 Mesylation of 16 and subsequent debenzylation using
transfer hydrogenolysis afforded mesylate 18 in 67% yield over
two steps. Coupling of tris(tetrabutylammonium) diphosphate to
18 was accomplished using the general procedure reported by
Davisson et al.15 and afforded epoxide analogue 10 in 40% yield.
measured by monitoring the consumption of reduced MV at 734
nm.9 As summarized in Table 1 and Figure 1c, 10 is a kinetically
competent IspG substrate exhibiting a kcat of 20.1 min-1, which is
very close to that of the natural IspG substrate MEcPP (6) (Figure
1b, Table 1). Interestingly, the Km for 10 (Km ) 119 ( 24.5 µM)
is ∼3-fold smaller than that of 6 (Km ) 311 ( 21.4 µM), and
apparent substrate inhibition was observed (Ki ) 1.3 ( 0.4 mM).
Our studies indicate that 10, an intermediate in the IspG reaction
mechanism proposed by Rohdich et al.,11 is catalytically competent
as an IspG substrate, and they provide the first direct experimental
evidence suggesting that an epoxide intermediate is possible in the
catalytic mechanism of this intriguing enzyme. The kinetic param-
eters of 10 relative to MEcPP suggest the possibility that IspG could
accommodate linear diphosphate 10 with an affinity comparable
to that for the structurally dissimilar MEcPP. The lower Km value
of 10 is consistent with an enzymatic conformational state along
the natural reaction pathway that has high affinity for the linear
diphosphate epoxide intermediate, to prevent nonproductive release
into solution. Furthermore, the similar kcat values suggest that the
natural substrate and the epoxide share a common rate-limiting step
that is post-epoxide formation, if the epoxide is indeed an
intermediate along the reaction pathway.
Acknowledgment. This work was supported by funding from
The Johns Hopkins Malaria Research Institute Pilot Grant (R.L.N.
and C.L.F.M.) and startup funds to P.L. and C.L.F.M. P.L. was
also supported by NSF CAREER Award (CHE-0748504).
Supporting Information Available: Procedures for the synthesis
of compound 10, characterization of the IspG-catalyzed enzymatic
product of 10, and optimization of IspG activity for compound 10. This
material is available free of charge via the Internet at http://pubs.acs.org.
Figure 1. (a) 1H NMR analysis of the IspG-catalyzed conversion of 10 to
HMBPP. Reaction conditions: 100 mM Tris buffer (pH 8.0) at 37 °C for
30 min with 5 mM dithionite, 1 mM MV, 1 mM 10, and various IspG
concentrations. (b) Michaelis-Menten analysis of MEcPP (6). (c)
Michaelis-Menten analysis of 10.
References
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Compound 10 was evaluated as an IspG substrate using our
recently reported 1H NMR assay.9 This assay monitors IspG activity
by detecting the C3′ methyl protons of MEcPP (1.26 ppm) and
HMBPP (1.54 ppm) (see the Supporting Information). The C3′
methyl proton of epoxide analogue 10 has a chemical shift of 1.22
ppm (Figure 1a, trace A). As shown in Figure 1, IspG catalyzes the
conversion of 10 to HMBPP (7) in an enzyme-dependent manner
(Figure 1a, traces B-E). The enzyme-generated product was purified
by HPLC and characterized using 1H NMR and high-resolution mass
spectrometry. The data are consistent with the production of HMBPP
as the product (see the Supporting Information).
Table 1. Kinetic Parameters for 10 and MEcPP (6)
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Arigoni, D.; Rohdich, F. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14837–
14842.
substrate
IspG (nM)
Km (µM)a
Vmax (µM/min)
kcat (min-1
)
Ki (mM)
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Gra¨wert, T.; Amslinger, S.; Eisenreich, W.; Bacher, A.; Arigoni, D. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 1586–1591.
MEcPP
10
200
400
311 ( 21
119 ( 25
4.7 ( 0.1
8.0 ( 0.9
23.7
20.1
n.d.
1.3 ( 0.4
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Wessjohann, L. A. ChemBioChem 2004, 5, 311–323.
(13) Itoh, T.; Nagano, T.; Sato, M.; Hirobe, M. Tetrahedron Lett. 1989, 30,
6387–6388.
a The following equations were used for MEcPP and 10, respectively:
V ) Vmax[S]/(Km + [S]) and V ) Vmax/(1 +Km/[S] + [S]/Ki).
(14) Fontana, A. J. Org. Chem. 2001, 66, 2506–2508.
(15) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremer, K. E.; Muehlbacher,
M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768–4779.
We further characterized the epoxide analogue 10 by kinetic
analysis (Figure 1c) using dithionite as the reducing agent and
methyl viologen (MV) as the redox mediator.9 Initial velocities were
JA907470N
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