Stereochemical analysis of isopentenyl diphosphate isomerase type II from Staphylococcus aureus using chemically synthesized (S)- and (R)-[2- 2H]Isopentenyl diphosphates

Article abstract of DOI:10.1021/ol0524050

(Chemical Equation Presented) To study the catalysis of isopentenyl diphosphate (IPP) isomerase type II from Staphylococcus aureus, which is a flavoprotein catalyzing the interconversion of IPP and dimethylallyl diphosphate, we have chemically synthesized (S)- and (R)-[2-²H]IPP and carried out stereochemical analysis of the reaction. Our results show that the C-2 deprotonation of IPP by this enzyme is pro-R stereospecific, suggesting a similar stereochemical course as the type I enzyme.

Full text of DOI:10.1021/ol0524050

ORGANIC
LETTERS
2005
Vol. 7, No. 25
5677-5680
Stereochemical Analysis of Isopentenyl
Diphosphate Isomerase Type II from
Staphylococcus aureus Using
Chemically Synthesized (S)- and
(R)-[2-2H]Isopentenyl Diphosphates
Chai-lin Kao,† William Kittleman,† Hua Zhang,† Haruo Seto,‡ and Hung-wen Liu*,†
DiVision of Medicinal Chemistry, College of Pharmacy and Department of Chemistry
and Biochemistry, UniVersity of Texas at Austin, Austin, Texas 78712, and Department
of Applied Biology and Chemistry, Faculty of Applied Bio-Science, Tokyo UniVersity of
Agriculture, Tokyo 156-8502, Japan
liu@mail.utexas.edu
Received October 4, 2005
ABSTRACT
To study the catalysis of isopentenyl diphosphate (IPP) isomerase type II from Staphylococcus aureus, which is a flavoprotein catalyzing the
interconversion of IPP and dimethylallyl diphosphate, we have chemically synthesized (S)- and (R)-[2-2H]IPP and carried out stereochemical
analysis of the reaction. Our results show that the C-2 deprotonation of IPP by this enzyme is pro-R stereospecific, suggesting a similar
stereochemical course as the type I enzyme.
Isoprenoids, such as steroids, terpenoids, carotenoids, and
ubiquinones, play important roles in all living organisms.1
The basic building blocks for assembling these compounds
are two 5-C precursors, isopentenyl diphosphate (IPP, 1) and
dimethylallyl diphosphate (DMAPP, 2). In eukaryotic organ-
isms IPP is the primary product of the mevaloante (3)
pathway,2 and DMAPP is derived from IPP by the action of
IPP isomerase (Scheme 1, route A).3 In contrast, both IPP
and DMAPP are produced from 1-deoxy-D-xylulose (4) in
most bacteria and green algae,4 where IPP isomerase, if it is
present, may play a role to fine-tune the ratio of 1 and 2 to
meet the specific needs for the downstream processes
(Scheme 1, route B).5
Two types of IPP isomerase are known. The type I
enzyme,6 which requires only divalent metal ions for activity,
catalyzes isomerization via a carbocation intermediate and
Scheme 1
† Univeristy of Texas at Austin.
‡ Tokyo University of Agriculture.
(1) Scchettini, J. C.; Poulter, C. D. Science 1997, 277, 1788-1789.
(2) Bochar, D. A.; Friesen, J. A.; Stauffacher, C. V.; Rodwell, V. W. In
ComprehesiVe Chemistry of Natural Products; Barton, D., Nakanishi, K.,
Meth-Cohn, O., Eds.; Pergamon: New York, 1999; Vol. 2, pp 15-44.
(3) Koyama, T.; Ogura, K. In ComprehesiVe Chemistry of Natural
Products; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Pergamon: New
York, 1999; Vol. 2, pp 69-96.
10.1021/ol0524050 CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/09/2005

has been extensively characterized.7 In contrast, the more
recently discovered type II isomerase5,8 is a flavoprotein
whose activity requires divalent metal ion and NAD(P)H.9
Since the cofactor requirements for these two types of IPP
isomerase are so different, the type II isomerase may operate
by a mechanism distinct from that of the type I enzyme.10
Thus far, no mechanistic information about the type II
isomerase has been reported,11 presenting an exciting op-
portunity to initiate an investigation into this unusual enzyme.
In addition, the fact that the type II isomerase is essential
for some pathogens, including multidrug-resistant strains of
Staphylococcus aureus, Streptococci, and Enterococci, whereas
only type I isomerase is used in mammals, makes it an
attractive target for therapeutic agents.4b,5,8
by the yeast type I isomerase.14 However, the actual
stereospecificity of the C-2 deprotonation step of the Syn-
echocystis isomerase was not determined.
As part of our efforts to investigate the mechanism of type-
II isomerase, we have recently expressed the gene for the
type II isomerase from Staphylococcus aureus in Escherchia
coli according to the reported protocols8a and carried out a
stereochemical analysis of the reaction. Reported herein are
the results, which clearly indicate that the C-2 deprotonation
of IPP by the Staphylococcus isomerase is pro-R stereospe-
cific, suggesting an analogous stereochemical course as the
type I isomerase.
To facilitate the analysis, a new and convenient chemical
synthesis for preparing the stereospecifically labeled (S)-[2-
2H]IPP (5) and (R)-[2-2H]IPP (6) was developed.15 As
depicted in Scheme 2, the synthesis of (S)-[2-2H]IPP (5)
The reaction catalyzed by type I isomerase is a well-
established reversible 1,3-antarafacial process involving the
loss of the 2R-hydrogen of IPP and the addition of a solvent
hydrogen to C-4 of DMAPP.12 Although the mechanism of
type II isomerase remains elusive, the stereochemical course
of the deprotonation step for the type II enzyme from Bacillus
subtilis has been determined to be similar to that of type I
enzyme on the basis of an elegant, yet complex, labeling
study.13 In a separate report, prolonged incubation of IPP/
DMAPP with the type II isomerase from Synechocystis PCC
Scheme 2
2
6803 in H2O led to the incorporation of one deuterium at
C-2 and two deuteria at C-4 of IPP.14 Meanwhile, all three
hydrogens of the (E)-methyl group of DMAPP were ex-
changed with deuterium. Interestingly, under similar condi-
tions with the type I enzyme from yeast, only the protons at
C-1 of IPP and DMAPP remained unexchanged. It was thus
concluded that the reaction catalyzed by the Synechocystis
type II enzyme is more stereoselective than the one catalyzed
(4) (a) Rohdich, F.; Hecht, S.; Bacher, A. Pure Appl. Chem 2003, 75,
393-405. (b) Rohdich, F.; Bacher, A.; Eisenreich, W. Bioorg. Chem. 2004,
32, 292-308.
(5) Laupitz, R.; Hecht, S.; Amslinger, S.; Zepeck, F.; Kaiser, J.; Richter,
G.; Schramek, N.; Steinbacher, S.; Huber, R.; Arigoni, D.; Bacher, A.;
Eisenreich, W.; Rohdich, F. Eur. J. Biochem. 2004, 271, 2658-2669.
(6) (a) Rilling, H. C.; Bloch, K. J. Biol. Chem. 1959, 234, 1424-1432.
(b) Cornforth, J. W.; Popjak, G. Tetrahedron Lett. 1959, 19, 29-35.
(7) (a) Muehlbacher, M.; Poulter, C. D. Biochemistry 1988, 27, 7315-
7325. (b) Wouters, J.; Oudjama, Y.; Barkley, S. J.; Tricott, C.; Stalon, V.;
Droogmans, L.; Poulter, C. D. J. Biol. Chem. 2003, 278, 11903-11908.
(c) Wouters, J.; Oudjama, Y.; Stalon, V.; Droogmans, L.; Poulter, C. D.
Proteins: Struct., Funct., Bioinf. 2004, 54, 216-221.
(8) (a) Kaneda, K.; Kuzuyama, T.; Takagi, M.; Hayakawa, Y.; Seto, H.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 932-937. (b) Takagi, M.; Kaneda,
K.; Shimizu, T.; Hayakawa, Y.; Seto, H.; Kuzuyama, T. Biosci. Biotechnol.
Biochem. 2004, 68, 132-137. (c) Barkely, S. J.; Cornish, R. M.; Poulter,
C. D. J. Bacteriol. 2004, 186, 1811-1817. (d) Yamashita, S.; Hemmi, H.;
Ikeda, Y.; Nakayama, T.; Nishino, T. Eur. J. Biochem. 2004, 271, 1087-
1093.
(9) Under aerobic conditions, NADPH is required for enzyme activity.
However, if purified and assayed under strictly anaerobic conditions, the
addition of NADPH is no longer necessary.5
(10) The crystal structure of the type II isomerase from Bacillus subtilis
was recently determined; however, the substrate (1/2) binding site could
not be clearly defined (Steinbacher, S.; Kaiser, J.; Gerhardt, S.; Eisenreich,
W.; Huber, R.; Bacher, A.; Rohdich, F. J. Mol. Biol. 2003, 329, 973-
982).
begins with (2R)-trans-3-methyloxiranemethanol (8), which
was the product of Sharpless epoxidation of crotyl alcohol
(15) Compounds 5 and 6 were traditionally prepared enzymatically from
labeled mevalonic acid (see: Cornforth, J. W.; Cornforth, R. H.; Donninger,
C. Popjak, G. Proc. R. Soc. London, Ser. B 1965, 163, 492-514. Sagami,
H.; Ogura, K.; Seto, S. Biochemistry 1977, 16, 4616-4622). They could
also be chemically synthesized from dimethylally alcohol (Suga, T.; Ohta,
S.; Ohmoto, T. Chem. Soc., Perkin Trans. 1 1987, 2845-2848). A synthesis
of 6 starting from D-mannitol 1,2:5,6-bis-acetonide in high enantiomeric
excess was recently reported (Leyes, A. E.; Poulter, C. D. Org. Lett. 1999,
1, 1067-1070).
(11) Bornemann, S. Nat. Prod. Rep. 2002, 19, 761-772.
(12) (a) Clifford, K.; Cornforth, J. W.; Mallaby, R.; Philips, G. T. Chem.
Commun. 1971, 1599-1600. (b) Cornforth, J. W.; Clifford, K.; Mallaby,
R.; Philips, G. T. Proc. R. Soc. London, Ser. B. 1972, 182, 277-295.
(13) Laupitz, R.; Grawert, T.; Rieder, C.; Zepeck, F.; Bacher, A.; Arigoni,
D.; Rohdich, F.; Eisenreich, W. Chem. BiodiVersity 2004, 1, 1367-1376.
(14) Barkley, S. J.; Desai, S. B.; Poulter, C. D. Org. Lett. 2004, 6, 5019-
5021.
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Org. Lett., Vol. 7, No. 25, 2005

(7) using (+)-diisopropyltartarate as a catalyst.16 The enan-
tiomeric purity of 8 was estimated to be greater than 95%
by the comparison of its optical rotation data with the
reported value.16 Silyl protection of the hydroxyl group
followed by reduction of 9 by lithium triethylborohydride
(super hydride) led to the formation of 10a as the sole
product. To our surprise, an opposite regiospecific ring
opening was observed when the reduction of 9 was carried
out using lithium triethylborodeuterride (super deuteride), in
which compound 10c emerged as the only identifiable
product (58% yield). Interestingly, the regioselectivity was
sensitive to the presence of Lewis acid in the reaction
mixture. An extensive survey of different conditions led to
the finding that an 11:1 product ratio of 10b and 10c could
be achieved when the reaction was carried out with a
stoichiometric amount of anhydrous MgCl2.
1 mM dithiothreitol (DTT), and 10 µM of enzyme in 1 mL
of 100 mM potassium phosphate buffer (pH 7.0) was
prepared. The reaction was incubated at 37 °C for 20 h and
stopped by rapid freezing in liquid nitrogen. The samples
were lyophilized, redissolved in D2O, and lyophilized again.
The dry residue was then dissolved in D2O and subjected to
NMR analysis. The sample derived from (R)-[2-2H]IPP (6)
shows two methyl singlets at δ 1.56 (3H, s) and 1.61 (3H,
s), an olefinic proton resonance at δ 5.30 (1H, m), and a
methylene signal at δ 4.31 (2H, t, J ) 7 Hz) (Figure 1, top),
Although the byproduct 10c could be removed by silica
gel chromatography, the isolated yield of 10b was low as a
result of its high volatility. Thus, to avoid the unnecessary
loss of 10b, the mixture of 10b and 10c was treated with
oxidizing agents without purification to make the corre-
sponding keto products. Among the various oxidants tested,
including Dess-Martin periodant, O-iodoxybenzoic acid
(IBX), RuO4, and N-methylmorpholine N-oxide (NMO)-
tetrapropylammonium perruthenate (TPAP), NMO-TPAP,
was found to be most effective. However, the pretreatment
of the reaction mixture with excess NMO was crucial for
consistent results. In addition to its co-oxidant role, this
reagent may serve as a scavenger to neutralize the residual
reducing species remaining from the previous step. The
resulting 3-keto and 2-keto products (11b and 11c, respec-
tively) were separated, and compound 11b was subjected to
olefination under Wittig conditions to give tert-butyl-
dimethyl-((2S)-3-methyl-[2-2H]-but-3-enyloxy)-silane (12b)
in 45% yield.
Deprotection of 12b by anhydrous tetrabutylammonium
fluoride (TBAF) followed by tosylation of the isopentenyl
alcohol product afforded 13b in 95% yield. The remaining
moisture from the deprotection reaction was found to
complicate the subsequent tosylation, so that the inclusion
of anhydrous CaH2 in the tosylation step was important to
ensure a complete transformation. The final pyrophospho-
rylation step to generate 5 was accomplished based on a
literature procedure17 in 51% yield. The (R)-[2-2H]IPP (6)
was prepared from the corresponding 2S-isomer of 8 (i.e.,
14) in an analogous manner.18
Figure 1. 1H NMR spectra showing the C-1 methylene and C-2
olefinic region of DMAPP generated in the incubation of (R)-[2-
2H]IPP (6) (top) or (S)-[2-2H]IPP (5) (bottom) with the Staphylo-
coccus type II IPP isomerase.
which are identical to an authenic DMAPP spectrum.
In contrast, the spectrum of the sample derived from (S)-
isomer (5) exhibits signals at δ 1.59 (3H, s), 1.63 (3H, s),
and 4.30 (2H, d, J ) 5 Hz) (Figure 1, bottom). The
disappearance of the δ 5.30 signal and the corresponding
change of the δ 4.30 signal to a doublet clearly demonstrated
retention of the deuterium label at C-2 in this sample. On
the basis of these observations, it can be concluded that the
enzyme removes only the hydrogen resided at the Hre position
of C-2 of IPP during catalysis. A complementary experiment
was also performed with the type I IPP isomerase isolated
from E. coli.19 As expected, the same pro-R stereochemical
preference was noted for this enzyme. This observation also
confirms the stereochemical assignment of the isotope
labeling in 5 and 6.20
With these labeled IPPs in hand, we proceeded to
determine the stereospecificity of the C-2 deprotonation step
catalyzed by the Staphylococcus enzyme, which was purified
and established to be a flavoprotein as previously reported.8a
Analogous to other members of type II isomerases, NAD-
(P)H as well as a divalent metal ion are required for its
activity. Accordingly, the reaction mixture containing 10 mM
of labeled IPP, 1 mM NADPH, 10 µM FMN, 10 mM MgCl2,
This work is significant for two reasons. First, a convenient
chemical method for the preparation of stereospecifically
(19) The Escherichia coli type I isomerase gene was amplified from the
genomic DNA, cloned into pET24b(+) vector and expressed in E. coli BL21
under the induction by 0.5 mM isopropyl â-D-thiogalactoside (IPTG). The
C-terminal His6-tagged enzyme was purified to greater than 90% purity
using Ni-NTA chromatography. The assay mixture contained 10 mM labeled
IPP, 200 mM KCl, 20 mM MgCl2, 1 mM DTT, 5 µM isomerase in 1 mL
of 100 mM potassium phosphate buffer, pH 7. The workup procedure and
subsequent NMR analysis were the same as those used for type II enzyme.
(20) Attempt to determine the chiral purity of 5 and 6 in poly
γ-benylglutamate methylene chloride solution based on a published method14
was unsuccessful.
(16) Gao, Y.; Klunder, J. M.; Hanson, R. M.; Masamune, H.; Ko, S. Y.;
Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765-5780.
(17) Davisson, V. J.; Woodside, A. B.; Poulter, C. D. Methods Enzymol.
1985, 110, 130-144.
(18) See Supporting Information for details.
Org. Lett., Vol. 7, No. 25, 2005
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labeled IPP, which is the biosynthetic precursor for iso-
prenoids in all living organisms, was developed. The ready
availability of these labeled IPPs should find wide application
in stereochemical studies of enzymes involved in the
biosynthesis of a great variety of secondary metabolites.
Second, the stereoselectivity of C-2 deprotonation catalyzed
by the Staphylococcus IPP isomerase has been unambigu-
ously determined to be pro-R specific, a preference identical
to its counterpart from Bacillus subtilis. The pro-R stereo-
specificity is conserved among all type I and type II IPP
isomerases studied thus far. Clearly, despite the different
cofactor requirements for these two classes of IPP isomer-
ases, the active sites of type II isomerases likely share
features with those of the type I enzymes in controlling the
orientation of the enzyme-substrate complex. Experiments
are in progress to explore the implications of these results
on the mechanism of this unusual enzyme.
Acknowledgment. This work was supported by the
Welch Foundation grant F-1511.
Supporting Information Available: Experimental pro-
cedures for the preparation of stereospecifically labeled 5
and 6. This material is available free of charge via the Internet
at http://pubs.acs.org.
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