The stereochemical course and mechanism of the IspH reaction

Article abstract of DOI:10.1002/anie.201201110

On the right path: The stereochemcial course of the IspH reaction, the last reaction in the deoxyxylulose phosphate pathway to terpenes, was investigated in feeding experiments with deuterated isotopologues of 1-deoxy-D-xylulose. The results support an enzyme mechanism for IspH that involves a previously suggested metallacyclopropane intermediate. Copyright

Full text of DOI:10.1002/anie.201201110

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DOI: 10.1002/anie.201201110
Terpene Biosynthesis
The Stereochemical Course and Mechanism of the IspH Reaction**
Christian A. Citron, Nelson L. Brock, Patrick Rabe, and Jeroen S. Dickschat*
Two pathways exist for the biosynthesis of the terpene
monomers dimethylallyl diphosphate (DMAPP) and isopen-
tenyl diphosphate (IPP): the mevalonate pathway[1] and the
deoxyxylulose phosphate (DOX) pathway.[2,3] Many patho-
genic bacteria and the malaria parasite Plasmodium falcipa-
rum exclusively use the DOX pathway, which is essential in
these organisms.[4,5] Since this pathway is not present in
humans, its enzymes represent attractive targets for new
antimicrobial drugs.[6] Therefore, a detailed understanding of
the enzyme mechanisms is of high interest.
hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP, 7)
into an approximate 1:5 mixture of DMAPP (8) and IPP (9)
has attracted considerable interest and is still under discus-
sion. The crystal structure of IspH showed a central Fe3S4
cluster,[8] while other data were in favor of an Fe4S4 cluster.[9]
Recently, the crystal structure of IspH in the presence of its
substrate 7 was refined to a structure with an Fe4S4 cluster.[10]
The fourth labile iron center binds the substrate 7,[9d,10] as well
as potent inhibitors such as pyridine diphosphates and
alkynes.[11] Mçssbauer parameters of the substrate-free
enzyme suggested that this Fe center is coordinated by
three S and two or three other ligands (O or N) that dissociate
upon substrate binding.[9b,d]
The intermediates and enzymes of the DOX pathway
have all been identified (Scheme 1).[7] In particular, the
unique mechanism of IspH that catalyzes the conversion of 1-
Three mechanisms have been proposed for the IspH
reaction (Scheme 2). Mechanism A, suggested by Rohdich
et al. (Scheme 2A), resembles a Birch reduction of complex
10 by a one-electron transfer, protonation, and elimination of
water to the allyl radical 11, and a second electron transfer to
give the allyl anion 12. Its protonation at C-2 or C-4 results in
IPP and DMAPP, respectively.[12] This mechanism is sup-
ported by isotopic labeling experiments with substrate
analogues.[13] Mechanism B, proposed by Wang et al. (Sche-
me 2B), is based on the ENDOR spectroscopic detection of
a paramagnetic intermediate trapped in the unreactive
E126A mutant that was interpreted as a metallacyclopropane
species.[14] Alternatively, this intermediate may also be
described as an h2-alkenyl p complex. The delineated mech-
anism proceeds from 10 by one-electron reduction to form the
h2-alkenyl/metallacycle 13 followed by a protonation/dehy-
dration to give the h1-allyl complex 14. A second electron
transfer yields 12 as the precursor for IPP and DMAPP.
Mechanism C was proposed by Altincicek et al. and includes
the initial protonation/dehydration of 10 assisted by the Lewis
acidity of the metal center to generate the allyl cation 15,
which undergoes two reduction steps via 11 to 12 and
a subsequent protonation to afford IPP and DMAPP.[15]
This mechanism is contradicted by the minor effect of
electron-withdrawing substituents in substrate analogues.[16]
Common intermediates of all three mechanisms are the
initial complex 10 and the allyl anion 12. The product ratio of
IPP and DMAPP was suggested to be controlled by the
proton transfer from the terminal phosphate group of
HMBPP, where the two oxygen atoms are at a distance of
3.4–3.5 ꢀ to C-1 and C-3 (Scheme 2A).[10,12b] This hypothesis
is in full agreement with the observed stereospecific C-3
protonation of HMBPP from the Si face.[17] The stereochem-
ical course of almost all the steps of the DOX pathway have
been studied.[18] Along this pathway, the C-3 hydrogen atom
of 1 (HA, Scheme 1) becomes the aldehyde hydrogen atom in
2 and ends up as the pro-S hydrogen atom at C-1 in 3, whereas
the pro-R hydrogen atom (HC) is introduced by NADPH.[19]
All subsequent transformations from 3 to 6 proceed with an
Scheme 1. Stereochemistry of the DOX pathway. CTP=cytidine tri-
phosphate, ATP=adenosine triphosphate, ADP=adenosine diphos-
phate, CMP=cytidine monophosphate.
[*] C. A. Citron, N. L. Brock, P. Rabe, Dr. J. S. Dickschat
Institut fꢀr Organische Chemie
Technische Universitꢁt Braunschweig
Hagenring 30, 38106 Braunschweig (Germany)
E-mail: j.dickschat@tu-bs.de
[**] This work was funded by the Deutsche Forschungs-gemeinschaft
(DFG) by an Emmy-Noether Fellowship (to J.S.D.) and the Fonds
der Chemischen Industrie with a scholarship (to N.L.B.). We thank
Prof. Dr. Stefan Schulz for excellent support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201201110.
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stereochemcial course can be made for mechanisms A and C:
The OH group of HMBPP remains bound to Fe up to the step
that determines the stereochemical outcome of the reaction.
This should result in HA being directed to the Z position and
of HC to the E position. In mechanism B, this OH group is
released in 13, thereby allowing rotation of the CH2OH
group. Consequently, no prediction of the stereochemical
outcomes of HA and HC is possible. Protonation at C-1 delivers
a potentially chiral methyl group in DMAPP with unknown
configuration, but the absolute configuration shown in
Scheme 2A should result from mechanisms A and C. We
report here on the stereochemical course of the IspH reaction
and its implications for the mechanism of the enzyme.
Deuterated isotopologues of 1-deoxy-d-xylulose, phos-
phorylated intracellularly to yield 1, were used in feeding
experiments to investigate the stereochemical course of the
IspH reaction. Deuterium labeling of HA can, in particular,
give important insights. A suitable system to follow the
deuterium labeling is pentalenene biosynthesis in streptomy-
cetes, since the detailed stereochemical course of all the
transformations to this sesquiterpene are known.
The reaction of DMAPP or any advanced polyisoprenoid
diphosphate such as geranyl diphosphate (GPP, 16) proceeds
with attack of IPP at C-4 from its Si face[22] and abstraction of
the pro-R hydrogen atom (Scheme 3A).[23] Thus, the terminal
olefinic HE of IPP ends up in the 4-pro-S position and HZ is
Scheme 2. Proposed mechanisms for the IspH reaction.
unchanged carbon backbone. Different mechanisms have
been suggested for the transformation of 6 to HMBPP,[20] but
it is well-established that C-2 and C-3 are deoxygenated and
HB is retained at C-3.[21] Only the stereochemical course of the
IspH reaction has not been fully established: It is not known
whether HA or HC in HMBPP ends up as the terminal olefinic
(E)- or (Z)-hydrogen atom in IPP, a fate that is predetermined
in intermediate 12 (Scheme 2). However, a prediction of the
Scheme 3. Stereochemical course of A) polyisoprenoid biosynthesis,
B) pentalenene biosynthesis, and C) the IDI reaction.
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2
found as the 4-pro-R proton. The conversion of farnesyl
diphosphate (FPP, 17) into pentalenene (23, Scheme 3B) is
initiated by the cyclization to the humulyl cation (18). Specific
removal of the 9-pro-S hydrogen atom yields a-humulene
(19). The abstracted proton is reintroduced at C-10 without
significant exchange with the medium.[24] Concomittant
cyclization to 20, a 1,2-Hꢀ shift to 21, cyclization to 22, and
deprotonation yields 23. The final deprotonation proceeds
with specific loss of the pro-R hydrogen atom,[25] which can be
traced back to HE in IPP. Therefore, the conversion of HA in
1 into either HE or HZ in IPP in the IspH reaction can be
followed by its fate in this step.
A series of deuterated isotopologues of 1-deoxy-d-xylu-
lose (30) were synthesized for the feeding experiments
(Scheme 4). All of these compounds were fed to Streptomyces
avermitilis and the incorporation of deuterium into 23 was
analyzed by GC-MS. An advantage of this analytical method
is the possibility to separate the deuterated from unlabeled
isotopologues gaschromatographically (see Figure S1 in the
Supporting Information).[26] Since this effect becomes more
significant as the deuterium content of the analyte increases,
the methyl groups in all of the synthesized isotopologues of 30
were deuterated.
of deuterium. The reaction with H3CMgI resulted in 27a,
which afforded 28a after oxidation with IBX. Sharpless
dihydroxylation with AD-mix b and catalytic hydrogenation
furnished 30a. For the synthesis of 30b, alkyne 24a was
reduced with LiAlH4 and then quenched with 2H2O, while 30c
was obtained from deuterated 24b, which is available from
tert-butyldimethylsilyl-protected propargylic alcohol (see the
Supporting Information). The other isotopologues 30d–h
(Scheme 4B) were all obtained by the same route by using the
appropriate combination of unlabeled and deuterated
reagents.
Feeding of 30a afforded [2H11]-23 (see Figure S2A,B in
the Supporting Information). The biosynthesis of [4,5,5,5-
2H4]DMAPP and [4,5,5,5-2H4]IPP can be expected from 30a
(HA = 2H). Since only 11 of the 12 deuterium atoms in the
three isoprene units of 23 are retained and one is lost, this
experiment clearly demonstrates that the stereochemical
course of the IspH reaction directs HA in 10 into the
E position and HC into the Z position of IPP (Scheme 5).
Although no direct evidence is obtained, the potentially chiral
ꢀ
methyl group CHAHBHD in DMAPP may have the absolute
configuration shown.
A flexible route for the introduction of deuterium into 30
was developed based on a procedure by Giner (Sche-
me 4A).[27] But-2-yn-1,4-diol monobenzyl ether (24a) was
treated with LiAl2H4 and then quenched with water to obtain
25a. Oxidation with IBX gave 26a without any detectable loss
Scheme 5. Stereochemical course of the IspH reaction.
Feeding of 30b resulted in [2H10]-23 (Figure S2C in the
Supporting Information). This is fully consistent with a con-
version of 30b into [2,5,5,5-2H4]DMAPP and (2R)-[2,5,5,5-
2H4]IPP (HB = 2H) as well as the formation of [2H10]FPP by
a loss of two deuterium atoms (HB) from the IPP elongation
units during the biosynthesis of polyisoprenoid. These data
reinforce the previously observed stereospecific C-3 proto-
nation of HMBPP from the Si face. The reason for some loss
of an additional deuterium, as indicated by the ion at m/z 213,
is the partial isomerization of (2R)-[2,5,5,5-2H4]IPP to [5,5,5-
2H4]DMAPP by isopentenyl diphosphate isomerase (IDI,
Scheme 3C), a reaction that specifically removes the pro-R
hydrogen atom (HB) for both type I and type II IDI,[17,23a,28]
and its incorporation into 23. A type I IDI, but no type II
enzyme, is encoded in the genome of S. avermitilis.
Feeding of 30c resulted in [2H15]-23 (Figure S2D in the
Supporting Information), with retention of all 15 deuterium
atoms. This result corroborates the previous finding that the
conversion of cation 18 via 19 into 20 (Scheme 3B) proceeds
without proton exchange with the medium, in full agreement
Scheme 4. Synthesis of deuterated 1-deoxy-d-xylulose. IBX=1-hydroxy-
1,2-benziodoxol-3(1H)-one 1-oxide, DMSO=dimethyl sulfoxide.
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with the known mechanism of the pentalenene synthase.[24,25]
Feeding experiments with the isotopologues 30d–h confirmed
the results obtained with 30a–c (see the Supporting Informa-
tion).
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The delineated stereochemical course of the IspH reac-
tion (Scheme 5) has some important implications on the
enzyme mechanism (Scheme 2). As outlined above, it is
difficult to understand the stereochemical course of HA!HE
and HC!HZ through mechanisms A and C, whereas in
mechanism B the OH group of HMBPP is released from
the Fe center in 13. This allows rotation of the CH2OH group
into a conformation that readily explains the observations.
Interestingly, such a conformation was recently described by
Glide docking calculations and molecular mechanics optimi-
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crystal structures of several IspH mutants.[29] In summary, the
stereochemical course of the IspH reaction is consistent with
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In conclusion, we have presented the stereochemical
course of the IspH reaction giving important insights in the
enzyme mechanism. This will allow for the precise local-
ization of deuterium in feeding experiments concerning the
biosynthesis of terpenes made through the DOX pathway.
The consequences of the mechanism of IspH may assist in the
rational design of new IspH inhibitors as potential drugs.
Received: February 9, 2012
Published online: March 12, 2012
Keywords: biosynthesis · DOX pathway · ISPH enzyme ·
.
stereochemistry · terpenes
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