Study of IspH, a key enzyme in the methylerythritol phosphate pathway using fluoro-substituted substrate analogues

Article abstract of DOI:10.1021/ol202559r

IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies

Full text of DOI:10.1021/ol202559r

ORGANIC
LETTERS
2011
Vol. 13, No. 21
5912–5915
Study of IspH, a Key Enzyme in the
Methylerythritol Phosphate Pathway Using
Fluoro-Substituted Substrate Analogues
Youli Xiao,† Wei-chen Chang,‡ Hung-wen Liu,*,‡ and Pinghua Liu*,†
Department of Chemistry, Boston University, Boston, Massachusetts 02215,
United States, and Division of Medicinal Chemistry, College of Pharmacy, and
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin,
Texas 78712, United States
h.w.liu@mail.utexas.edu; pinghua@bu.edu
Received September 21, 2011
ABSTRACT
IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies of IspH using fluoro-
substituted substrate analogues to dissect the contributions of several factors to IspH catalysis, including the coordination of the HMBPP C4ꢀOH
group to the ironꢀsulfur cluster, the H-bonding network in the active site, and the electronic properties of the substrates, are reported.
IspH is a [4Fe-4S]-cluster-containing enzyme found in
the newly discovered isoprene biosynthetic pathway known
as the methylerythritol phosphate (MEP) pathway.1 It
catalyzes the last step of the MEP pathway by reductive
dehydroxylation of the C4ꢀOH group of 4-hydroxy-3-
methyl-butenyl diphosphate (HMBPP, 1) to isopentenyl
diphosphate (IPP, 2) and dimethylallyl diphosphate
(DMAPP, 3, Figure 1), which are the two building blocks
for all isoprenoids.2,3 While the MEP pathway is not
present in humans, it is essential for many pathogenic
microorganisms. Therefore, the enzymes involved in this
pathway are attractive new targets for antimicrobial
drugs.3,4 Early results showed that its labile [4Fe-4S]
cluster contains a unique iron site,5ꢀ10 to which the
C4ꢀOH group of HMBPP binds (4 in Scheme 1).6,7,11 The
bound HMBPP adopts a hairpin conformation in the active
(5) Wolff, M.; Seemann, M.; Tse Sum Bui, B.; Frapart, Y.; Tritsch,
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(8) Grawert, T.; Rohdich, F.; Span, I.; Bacher, A.; Eisenreich, W.;
Eppinger, J.; Groll, M. Angew. Chem., Int. Ed. 2009, 48, 5756–5759.
(9) Rekittke, I.; Wiesner, J.; Rohrich, R.; Demmer, U.; Warkentin,
E.; Xu, W. Y.; Troschke, K.; Hintz, M.; No, J. H.; Duin, E. C.; Oldfield,
E.; Jomaa, H.; Ermler, U. J. Am. Chem. Soc. 2008, 130, 17206–17207.
† Boston University.
‡ University of Texas at Austin.
€
(10) Grawert, T.; Kaiser, J.; Zepeck, F.; Laupitz, R.; Hecht, S.;
(1) Rohmer, M. Nat. Prod. Rep. 1999, 16, 565–574.
(2) Eisenreich, W.; Bacher, A.; Arigoni, D.; Rohdich, F. Cell. Mol.
Life Sci. 2004, 61, 1401–1426.
(3) Thibodeaux, C. J.; Liu, H. W. Chimia 2009, 63, 334–339.
(4) Oldfield, E. Acc. Chem. Res. 2010, 43, 1216–1226.
Amslinger, S.; Schramek, N.; Schleicher, E.; Weber, S.; Haslbeck, M.;
Buchner, J.; Rieder, C.; Arigoni, D.; Bacher, A.; Eisenreich, W.;
Rohdich, F. J. Am. Chem. Soc. 2004, 126, 12847–12855.
(11) Xiao, Y.; Zhao, Z. K.; Liu, P. J. Am. Chem. Soc. 2008, 130, 2164–
2165.
r
10.1021/ol202559r
Published on Web 10/07/2011
2011 American Chemical Society

site with the carbon chain skeleton sandwiched between its
pyrophosphate group and the [4Fe-4S] cluster (Figure 1).12
Due to the lack of a nearby proton source, protonation of
the allylic anion intermediate (8, Scheme 1) during turn-
over has been proposed to be mediated by the terminal
phosphate group of HMBPP.8,11,12
metallo-center may facilitate the heterolytic cleavage of the
C4ꢀOH bond to form an allylic cation intermediate (12),
which then undergoes two-electron reduction and proto-
nation to yield the final products. Although coordination
of the C4ꢀOH group of HMBPP to the [4Feꢀ4S] cluster is
important for all three proposed mechanisms, formation
of metallocycle intermediates using the HMBPP olefinic
moiety is specific for mechanism B, and the involvement of
a cation intermediate is unique for mechanism C.
Scheme 1. Proposed Mechanisms for IspH-Catalyzed Reaction
Figure 1. Reaction catalyzed by IspH, and the IspH-HMBPP (1)
complex in the active site. Due to the short distance (∼ 2.9ꢀ
3.0A) between the olefinic moiety (C2ꢀC3) of 1 and the unique
apical iron site, some interactions between the double bond of 1
and the ironꢀsulfur cluster may exist.
Studies in recent years have led to three mechanistic
models for IspH catalysis.5,8,11,13ꢀ16 Mechanism A pro-
posed by Rohdich et al. (Scheme 1, route A) resembles a
Birch reduction in which a one-electron transfer from the
reduced ironꢀsulfur cluster to HMBPP (5 f 6) triggers the
C4-deoxygenation from the nascent radical anion species
(6) to form an allyl radical intermediate (7).11,14,15 Subse-
quent one-electron reduction (7 f 8) followed by proto-
nation produces IPP (2) or DMAPP (3) depending on
whether protonation occurs at C2 or C4 of 8. Mechanism B
proposed by Wang et al. (Scheme 1, route B) is based
mainly on the biophysical characterization of a paramag-
netic species trapped in the IspH E126A mutant.17 In this
organometallic model, formation of a substrate/metal π
complex or an η2-alkenyl/metallacycle (9) followed by
dehydration gives an η1-allyl intermediate (10). Upon the
second one-electron transfer, 10 is reduced to an η3-allyl
complex (11), which is then protonated to form IPP or
DMAPP. Alternatively, in mechanism C (Scheme 1, route
C) proposed by Altincicek et al.,13 the Lewis acidity of the
To learn more about the substrate specificity and to
assess the energetic contributions of various interactions in
the active site during IspH catalysis, a series of fluoro-
substituted substrate analogues were synthesized and their
competence as IspH substrates was examined (Scheme 2).18
The results and insights gained from these studies are
reported herein.
Previously, we reported that IspH can process the
monofluoro analogue 13 to produce both IPP (2) and
DMAPP (3) in a ratio of ∼5:1.11 The kinetics of 13 as an
€
(12) Grawert, T.; Span, I.; Eisenreich, W.; Rohdich, F.; Eppinger, J.;
Bacher, A.; Groll, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1077–
1081.
(13) Altincicek, B.; Duin, E. C.; Reichenberg, A.; Hedderich, R.;
Kollas, A. K.; Hintz, M.; Wagner, S.; Wiesner, J.; Beck, E.; Jomaa, H.
FEBS Lett. 2002, 532, 437–440.
(14) Rohdich, F.; Zepeck, F.; Adam, P.; Hecht, S.; Kaiser, J.;
€
(18) Syntheses of the substrate analogues listed in Scheme 2 are
included in the Supporting Information. These compounds were eval-
uated as IspH substrates by a recently developed assay.7 After incuba-
tion of substrate analogues with IspH, the reaction mixture was purified
by HPLC, and the turnover products were isolated and characterized by
1H NMR and high-resolution mass spectrometry. The identities of these
products were further confirmed by comparison with synthetic stan-
dards (see Supporting Information).
Laupitz, R.; Grawert, T.; Amslinger, S.; Eisenreich, W.; Bacher, A.;
Arigoni, D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1586–1591.
(15) Xiao, Y.; Liu, P. Angew. Chem., Int. Ed. 2008, 47, 9722–9725.
(16) Wang, W.; Wang, K.; Liu, Y. L.; No, J. H.; Li, J.; Nilges, M. J.;
Oldfield, E. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4522–4527.
(17) Wang, W.; Li, J.; Wang, K.; Huang, C.; Zhang, Y.; Oldfield, E.
Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 11189–11193.
Org. Lett., Vol. 13, No. 21, 2011
5913

the apical iron site of the ironꢀsulfur cluster and the
change in the electrophilic nature of the double bond.
Notably, comparing 13 with 14, the Km for 14 is 4.7-fold
higher, the kcat is 3.3-fold lower, and the kcat/Km is ∼15.5-
fold reduced. The observed activity differences between 13
and 14 suggest that the C4-fluoro group of 13 may still
maintain partial hydrogen-bond interaction with the T167
and E126 side chain rendering it a better substrate for IspH
than 14.
Scheme 2. Products Produced from Incubations of Substrate
Analogues with IspHa
To further probe the effects of the substrate electronic
properties on IspH catalysis, (Z)-4,4,4-trifluoro-3-(hydroxy-
methyl)-but-2-en-1-yl diphosphate (15) was prepared. The
strong electron-withdrawing nature of the trifluoromethyl
substituent will destabilize the proposed allylic carboca-
tionintermediate(12) whichwillinturnreducethe reaction
rate to a great extent if mechanism C is operative. When 15
was incubated with IspH, 3-(trifluoromethyl)but-3-en-1-yl
diphosphate (16) was produced as the only product (see
Scheme 3).18 Clearly, IspH is capable of catalyzing the
dehydroxylation of15, but not the subsequent dehalogena-
tion reaction. A similar outcome was also observed when
(Z)-4,4,4-trifluoro-3-methyl-but-2-en-1-yl diphosphate (17)
or (E)-4,4,4-trifluoro-3-methyl-but-2-en-1-yl diphosphate
(18) was incubated with IspH and no product could be
detected (Scheme 2). Although the kcat/Km value is reduced
∼150-fold for 15 (compared to 1), the kcat is only 6.6-fold
lower. These data suggest that route C is less likely as the
mechanism for IspH. A similar conclusion was also reached
in a recent report in which incubation of IspH with several
proposed carbocationic intermediates caused little inhibi-
tion against IspH.19 The fact that the kcat for 15 is ∼3.3-fold
greater than that of the monofluoro analogue 13 and ∼10.9-
fold higher than that of the monofluoro analogue 14 again
underscores the important role played by the C4 hydroxyl
group in IspH catalysis.
a Refer to Figure 1 for the distances between various atoms.
IspH substrate was re-examined in this work by a more
sensitive assay using methyl viologen, instead of flavodox-
in-flavodoxin reductase, as the electron mediator.7 In the
new assay, the IspH activity is nearly 100-fold higher than
that of the NADPH-flavodoxin-flavodoxin reductase
system.11 As listed in Table 1, the Km for 13 is 5.3-fold
higher and kcat is 21.8-fold lower with a total of ∼115-fold
reduction of kcat/Km relative to that of HMBPP (1).
Clearly, replacing the C4ꢀOH group by a fluorine sub-
stituent affects overall catalysis. The reduction in catalysis
may result from a combination of several factors. First,
substitution of the C4ꢀOH group by a fluorine atom will
dramatically reduce the interaction between the substrate
and the [4Feꢀ4S] cluster apical iron site because fluorine is
a poor metal ligand. Second, the electronegativity differ-
ence betweena fluoroand a hydroxyl group will changethe
electronic properties of the double bond, which, in turn,
may affect IspH activity. Third, the HMBPP C4 hydroxyl
group is part of the H-bonding network shown in Figure 1.
To dissect the relative contributions of the above factors to
IspH catalysis, four additional substrate analogues, 14, 15,
17, and 18, were prepared and examined (Scheme 3 and
Table 1).
The substrate analogue, 14, has the fluorine group at the
C5 position instead of C4. Such a change should have little
impact on the electronic properties of the olefin but will
affect the hydrogen-bond network with the active site
residues T167 and E126. Thus, a comparison with analo-
gue 13 may allow us to better assess the effect of the
hydrogen-bonding network in IspH catalysis. As shown
in Table 1, incubation of 14 with IspH showed that IspH
can accept 14 as a substrate and produce IPP (2) as the sole
product. In comparison with HMBPP (1), the Km for 14 is
25-fold higher, the kcat is 72-fold lower, and the kcat/Km is
∼1783-fold reduced. The significant reduction in kcat/Km
for 14 relative to 1 is not surprising and can be attributed to
a combination of the lack of direct coordination of 14 to
Table 1. Summary of Activity Using Various Mechanistic
Probes
probes
kcat (minꢀ1
)
Km (μM)
kcat/Km (μMꢀ1 minꢀ1
)
3
1
604 ( 17
27.7 ( 2.2
8.4 ( 1.9
91.6 ( 2.2
19.7 ( 2.4
104 ( 31
489 ( 170
447 ( 32
30.6
0.27
13
14
15
17
18
0.017
0.21
No detectable activity
No detectable activity
The fact that only IPP (2) is produced in the reaction
with 14 is intriguing since both IPP (2) and DMAPP (3) are
formed when HMBPP (1) is used as the substrate. As
shown in Scheme 3A, generation of 2 and 3 from 1 results
from protonation of the proposed allylic anion inter-
mediate 8 at C2 and C4, respectively. The proton donor is
(19) Wang, K.; Wang, W.; No, J. H.; Zhang, Y.; Zhang, Y.; Oldfield,
E. J. Am. Chem. Soc. 2010, 132, 6719–6727.
€
(20) Grawert, T.; Span, I.; Bacher, A.; Groll, M. Angew. Chem., Int.
Ed. 2010, 49, 8802–8809.
5914
Org. Lett., Vol. 13, No. 21, 2011

believed to be the terminal phosphate group of HMBPP,
which is within ∼3.4ꢀ3.5 A of the C2 and C4 position of
HMBPP (see Figure 1).12,20 In the case of 14, the negative
charge of the proposed allylic anion intermediate (e.g.,
19, Scheme 3B) is delocalized through C2, C3, and C5
instead of C2, C3, and C4 as shown in intermediate 8 (see
Scheme 3A). The fact that protonation takes place only at
C2, but not at C5, in 19 likely reflects the distance and
unfavorable orientation of C5 of 19 relative to the phos-
phate group (e.g., OB) in the active site (e.g., ∼4.6 A
between C5 and OB, Figure 1).
([5-13C]-20, Scheme 3C),21 which is expected to bind to
the [4Feꢀ4S]þ cluster in two different orientationsdepend-
ing on whether the reaction follows mechanism A or B. If
the C4ꢀOH of 20 is the anchor that positions the substrate
in the active site (21 in Scheme 3D), protonation mediated
by OB at C4 of the allylic anion intermediate (23, Scheme 3D,
route a) would yield [13C]-IPP (2b) as the product (also see
Figure 1). In contrast, if the reaction proceeds via an
η2-alkenyl intermediate (22 in Scheme 3D), protonation
of the allylic anion at the carbon closer to OB (now C5,
Scheme 3D, route b) should afford [13C]-IPP (2c). The
observation that 2b is the sole product after incubation
strongly suggests that the C4ꢀOH group, rather than the
olefinic π-system, plays the dominant role in orienting
the substrate in the active site and, thus, favors the Birch
reduction model. Together, these observations strongly
indicate that protonation of the allylic anion intermediate
(8, 19, or 23) occurs at C2 and/or C4, but not at C5.
Scheme 3. Mode of Protonation of the Allylic Anion Inter-
mediate Generated during Turnover
In conclusion, the results obtained from these studies
provide important insights into the mode of substrate
binding and the mechanism of IspH. First, in view of
the relatively minor influence of electron-withdrawing
substituents on the reaction rate (e.g., 15), a mechanism
involving a carbocationic intermediate/transition state is
less likely. Second, coordination of the C4ꢀOH group of
HMBPP to the unique iron site of the [4Feꢀ4S] cluster and
its involvement in the hydrogen-bond network (e.g., with
T167 and E126) is crucial for efficient catalysis (13/14
versus 1, and 15 versus 17). The adverse effects (both steric
and electronic) imposed by a trifluoromethyl substituent in
17 can be balanced by the introduction of a C4ꢀOH group
(15 versus 17). Third, protonation of the allylic anion
intermediate is regiospecific and takes place only at C2
and/or C4 (see 8 f 2/3, 19 f 2, and 23 f 2b), but not at C5
(Scheme 3). These findings support the assigned role of
the terminal phosphate group of HMBPP in mediating the
final protonation step due to its close proximity to the
C2 and C4 positions of the allylic anion intermediate
(8, Schemes 1 and 3A). More experiments are in pro-
gress to further investigate this interesting example of
an enzymatic reductive dehydroxylation reaction.
Acknowledgment. This work was supported in part by
grants from the National Institutes of Health (GM040541
to H.-w.L. and GM093903 to P.L.) and the Welch Foun-
dation (F-1511 to H.-w.L.) and NSF CAREER (CHE-
0748504) to P.L.
The conclusions reached based on the outcomes of these
fluoro-analogues are alsoconsistent withthatderivedfrom
the incubation of IspH with a [13C]-labeled substrate
analogue, 3-(hydroxymethyl)but-3-en-1-yl diphosphate
Supporting Information Available. Details regarding
experimental procedures, NMR characterization of the
synthetic products as well as the incubation reactions.
This material is available free of charge via the Internet
at http://pubs.acs.org.
(21) Chang, W-c; Xiao, Y.; Liu, H-w; Liu, P. Angew. Chem., Int. Ed.
2011, in press.
Org. Lett., Vol. 13, No. 21, 2011
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