Stereochemistry of the reduction step mediated by recombinant 1-deoxy-D-xylulose 5-phosphate isomeroreductase

Article abstract of DOI:10.1021/ol990839n

(equation presented) The stereochemistry of the 1-deoxy-D-xylulose 5-phosphate (DXP) isomeroreductase reduction step has been examined using the recombinant enzyme from Synechocystis sp. PCC6803. Using [3-²H]DXP and [4S-²H]NADPH, it has been determined that the C1 pro-S hydrogen in the 2-C-methyl-D-erythritol 4-phosphate product derives from C3 of DXP, indicating that hydride attack occurs on the re face of the intermediate aldehyde. The 4S-hydride from NADPH is delivered, assigning this enzyme as a class B dehydrogenase.

Full text of DOI:10.1021/ol990839n

ORGANIC
LETTERS
1999
Vol. 1, No. 6
921-923
Stereochemistry of the Reduction Step
Mediated by Recombinant
1-Deoxy-D-xylulose 5-Phosphate
Isomeroreductase
Philip J. Proteau,* Youn-Hi Woo, R. Thomas Williamson, and
Chanokporn Phaosiri
College of Pharmacy, Oregon State UniVersity, CorVallis, Oregon 97331-3507
Phil.Proteau@orst.edu
Received July 20, 1999
ABSTRACT
The stereochemistry of the 1-deoxy-D-xylulose 5-phosphate (DXP) isomeroreductase reduction step has been examined using the recombinant
enzyme from Synechocystis sp. PCC6803. Using [3-2H]DXP and [4S-2H]NADPH, it has been determined that the C1 pro-S hydrogen in the
2-C-methyl-D-erythritol 4-phosphate product derives from C3 of DXP, indicating that hydride attack occurs on the re face of the intermediate
aldehyde. The 4S-hydride from NADPH is delivered, assigning this enzyme as a class B dehydrogenase.
The methylerythritol phosphate (MEP) pathway1 to iso-
prenoids has been the focus of intense research since the
first report of this biosynthetic route in 1993.2 The genes
for the first two enzymes in the pathway have now been
reported,3,4 with the gene encoding the 1-deoxy-D-xylulose
5-phosphate isomeroreductase (DXR)5 the most recently
identified.4 This enzyme converts 1-deoxy-D-xylulose 5-phos-
phate (DXP, 1) to 2-C-methyl-D-erythritol 4-phosphate
(MEP, 2) by catalyzing a rearrangement of the carbon
backbone and a NADPH-dependent reduction at C1 of the
proposed intermediate 2-C-methylerythrose 4-phosphate
(Figure 1). The stereochemistry of this reductive step is the
focus of this Letter.
(1) The methylerythritol phosphate pathway, or MEP pathway, was
suggested as a standardized name at the 4th European Symposium on Plant
Isoprenoids, Barcelona, Spain, April 21-23, 1999. Prior names have
included non-mevalonate pathway, mevalonate-independent pathway, tri-
osephosphate/pyruvate pathway, deoxyxylulose pathway.
(2) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H. Biochem.
J. 1993, 295, 517-524.
(3) Sprenger, G. A.; Scho¨rken, U.; Wiegert, T.; Grolle, S.; DeGraaf, A.
A.; Taylor, S. V.; Begley, T. P.; Bringer-Meyer, S.; Sahm, H. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 12857-12862.
Figure 1. Conversion of DXP to MEP by DXP isomeroreductase.
(4) Takahashi, S.; Kuzuyama, T.; Watanabe, H.; Seto, H. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9879-9884.
(5) DXP isomeroreductase describes the order of enzymatic transforma-
tions, an isomerization, followed by a reduction. This name is presented as
an alternative to DXP reductoisomerase.4
The stereochemical issues that need to be addressed are
the following: (1) Which hydrogen at C1 of 2-C-methyl-
10.1021/ol990839n CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/31/1999

erythritol phosphate originates from C3 of DXP and which
derives from NADPH? (2) Delivery of hydride from NADPH
occurs on which face of the intermediate aldehydesre or
si? (3) Which hydride from NADPH is delivered in the
reduction (class A or class B dehydrogenase)?
of all five backbone protons, and this solvent was used for
further analysis. HSQC and HMBC experiments were used
1
to assign the H and 13C resonances.9 GNOESY and 1D
DPFGSE10 NOE experiments were utilized to assign the
diastereotopic groups in the molecule. The C1 hydrogens
appeared as a pair of doublets, one centered at 3.71 ppm
and the other at 3.95 ppm. Strong NOE correlations between
the 3.71 ppm proton, the C2 methyl group, and one of the
C5 isopropylidene methyls11 allowed us to assign all three
of these groups to the same face of the dioxolane ring. On
the basis of NOE experiments, the pro-S proton at C1 has a
shift of 3.95 ppm and the pro-R proton resonates at 3.71
ppm.
To provide a monodeuterated compound for the stereo-
chemical analysis, it was necessary to synthesize deoxy-
xylulose phosphate specifically deuterated at C3. This was
accomplished by modifying the synthesis of 3-2H-deoxy-
xylulose12 to allow for synthesis of the 5-phosphate from
the known intermediate 6 (Scheme 2). The labeled substrate
Our earlier work examining the MEP pathway in the
cyanobacterium Synechocystis6 prompted us to examine the
cyanobacterial isomeroreductase. The Synechocystis protein
sequence has 42% identity to the Escherichia coli DXR
sequence. A cosmid clone containing the dxr gene was
obtained from the Kazusa DNA Research Institute. The
desired gene was subcloned into pUC18, and unique site-
elimination mutagenesis was used to introduce BamH I and
Hind III sites at the 5′ and 3′ ends of the gene, respectively.
The gene was then ligated into a pBAD/His expression
vector, and expression upon arabinose induction in E. coli
provided the recombinant isomeroreductase with a N-terminal
6xHis sequence. Partial purification using Ni-NTA spin
columns (Qiagen) provided recombinant enzyme for stere-
ochemical studies. The enzyme was assayed by monitoring
the decrease in absorbance at 340 nm.4 The product of the
enzymatic reaction was converted to 2-C-methylerythritol
triacetate (3) by treating the crude mixture with alkaline
phosphatase, followed by acetylation with acetic anhydride
and pyridine (Scheme 1). The purified derivative was
Scheme 2a
1
analyzed by comparing GC/MS and H NMR data with an
authentic standard.
Scheme 1a
a
(a) CH3C(OCH3)2CH3, pTsOH, THF; (b) H2, Pd/C, EtOH; (c)
tetrazole, (BnO)2PNiPr2; (d) tBuOOH, CH2Cl2; (e) H2, Pd(OH)2/
C; (f) Dowex 50, H2O.
7 was incubated with recombinant isomeroreductase, provid-
ing monodeuterated MEP which was dephosphorylated with
alkaline phosphatase. The proteins from the mixture were
precipitated with ethanol, and the supernatant was concen-
trated and then treated with acetic anhydride and pyridine.
The conversion of methylerythritol to the triacetate provided
a derivative that was readily purified by flash chromatog-
raphy (40% EtOAc/hexanes). The triacetate was deprotected
with a basic ion-exchange resin and then converted to the
a (a) DXR; (b) alkaline phosphatase; (c) Ac2O, pyr; (d) Amberlite
IRA400 (OH-); (e) CH3C(OCH3)2CH3, pTsOH, THF.
1
bisacetonide (Scheme 1). The H NMR spectrum of this
To distinguish the pro-R and pro-S protons at C1 of MEP,
a derivative was required in which the two protons could be
unambiguously assigned. A derivative that potentially could
fulfill this requirement was the bisacetonide derivative.7
Unlabeled 2-C-methylerythritol (4) was prepared8 and con-
verted to the bisacetonide 5 with 2,2-dimethoxypropane and
toluenesulfonic acid. A survey of NMR solvents revealed
that the 1H NMR spectrum in acetone-d6 provided resolution
derivative showed a slightly broadened singlet at 3.69 ppm
(9) 1H NMR (acetone-d6): δ 4.09 (dd, J ) 7.0, 5.8 Hz, H3), 4.00 (dd,
J ) 8.7, 7.0 Hz, H4S), 3.95 (d, J ) 8.7 Hz, H1S), 3.84 (dd, J ) 8.7, 5.8
Hz, H4R), 3.71 (d, J ) 8.7 Hz, H1R), 1.35 (s, C6-CH3R), 1.310 (s, C5-
CH3S), 1.306 (s, C5-CH3R), 1.27 (s, C6-CH3S), 1.21 (s, C2-CH3). 13C NMR
(acetone-d6): δ 110.11 (C5), 110.00 (C6), 81.83 (C2), 79.44 (C3), 73.49
(C1), 66.00 (C4), 27.58 (C5-CH3S), 27.02 (C5-CH3R), 26.60 (C6-CH3R),
25.04 (C6-CH3S), 19.80 (C2-CH3).
(10) GNOESY ) Gradient NOESY. Wagner, R.; Berger, S. J. Magn.
Reson. 1996, 123A, 229-232. DPFGSE ) double pulsed field gradient
spin-echo. Stott, K.; Keeler, J.; Van, Q. N.; Shaka, A. J. J. Magn. Reson.
1997, 125, 302-324.
(6) Proteau, P. J. Tetrahedron Lett. 1998, 39, 9373-9376.
(7) Anthonsen, T.; Hagen, S.; Sallam, M. A. E. Phytochemistry 1980,
19, 2375-2377.
(8) Duvold, T.; Cali, P.; Bravo, J. M.; Rohmer, M. Tetrahedron Lett.
1997, 38, 6181-6184.
(11) The selective excitation and high resolution possible with the
DPFGSE experiment allowed us to assign the correlations from the C2
methyl and the 3.71 ppm signal specifically to the methyl at 1.306 ppm.
(12) Giner, J.-L. Tetrahedron Lett. 1998, 39, 2479-2482.
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Org. Lett., Vol. 1, No. 6, 1999

[2H8]-2-propanol and alcohol dehydrogenase (NADP+-de-
pendent, from Thermoanaerobium brockii EC 1.1.1.2).16 The
deuterated NADPH samples were purified by HPLC.16
Independent incubations of these labeled NADPH samples
with unlabeled DXP and recombinant DXR provided MEP
that was subsequently dephosphorylated and converted to
the triacetate derivative for GC/EIMS analysis. An unlabeled
triacetate standard showed three key fragment ions at m/z
159, 129, and 117. The product from the [4S-2H]NADPH
incubation displayed fragment ions at 160, 129, and 118,
while the product from the 4R experiment had a mass
spectrum typical for the unlabeled standard. The increase in
mass by one unit for the m/z 159 and 117 ions demonstrates
the incorporation of a deuterium atom only when the
[4S-2H]NADPH is used. Transfer of the 4S-hydride classifies
DXP isomeroreductase as a class B dehydrogenase. Ketol-
acid reductoisomerase, a mechanistically related enzyme
involved in valine and isoleucine biosynthesis, is also a class
B dehydrogenase.17
Figure 2. Partial 1H NMR spectrum of 2-C-methylerythritol
bisacetonide. (A) Unlabeled standard. (B) Monodeuterated product
derived from enzymatic reaction.
while the doublet at 3.95 ppm is not present (Figure 2). This
result indicates that the proton from C3 of deoxyxylulose
becomes the pro-S proton of 2-C-methylerythritol.13 The
pro-R hydrogen at C1 of MEP, therefore, derives from
NADPH. This result also establishes that hydride delivery
is to the re face of the proposed aldehyde intermediate.
In addition to identifying the pro-S and pro-R hydrogens
in the bisacetonide derivative, the stereochemical assignments
for the triacetate methylene protons at C1 were determined.
The pro-S hydrogen resonates at 4.156 ppm, while the pro-R
hydrogen signal is at 3.90 ppm.14 Because the triacetate is a
readily accessible derivative from crude methylerythritol,
future efforts to determine the stereochemistry at C1 of MEP
could utilize the triacetate.
These experiments have demonstrated for the deoxy-
xylulose phosphate isomeroreductase from Synechocystis that
the C1 pro-S hydrogen derives from C3 of DXP. The pro-R
hydrogen, therefore, arises from NADPH. Delivery of
hydride is to the re face of the aldehyde with the pro-S
hydride from NADPH being transferred. (Figure 3).
The remaining stereochemical issue involved in this
transformation is which hydride is delivered from NADPH.
On the basis of incubation experiments with [1-2H]glucose
in the cyanobacterium Synechocystis sp., we have proposed
that the 4S-hydride of NADPH is delivered.6
Stereospecifically deuterated [4S-2H]NADPH was gener-
ated from NADP+ using glucose dehydrogenase (NADP+-
dependent, from Cryptococcus uniguttulatus EC 1.1.1.119)
and [1-2H]glucose.15 The [4R-2H]NADPH was prepared from
Figure 3. Stereochemical course for DXR-mediated conversion
of DXP to MEP.
Acknowledgment. This work was supported by the
Medical Research Foundation of Oregon. NMR spectra were
recorded on Bruker AC300 (NIH RR 04039 and NSF CHE-
8712343) and Bruker DRX600 (NSF CHE-9413692 and the
Keck Foundation) spectrometers. Dr. Satoshi Tabata of the
Kazusa DNA Research Institute is thanked for supplying the
cosmid clone containing the dxr gene.
(13) During the preparation of this manuscript, an article was published
in which a similar approach was used to address the stereochemistry of the
reduction step in the formation of 2-C-methylerythritol in higher plants:
Arigoni, D.; Giner, J.-L.; Sagner, S.; Wungsintaweekul, J.; Zenk, M. H.;
Kis, K.; Bacher, A.; Eisenreich, W. Chem. Commun. 1999, 1127-1128.
(14) 1H NMR (CDCl3): δ 5.18 (dd, J ) 8.0, 2.8 Hz, H3), 4.56 (dd, J )
11.8, 2.8 Hz, H4a), 4.163 (dd, J ) 11.8, 8.0 Hz, H4b), 4.156 (d, J ) 11.7
Hz, H1S), 3.90 (d, J ) 11.7 Hz, H1R), 2.11 (s, 3H), 2.09 (s, 3H), 2.05 (s,
3H), 1.24 (s, C2-CH3).
OL990839N
(16) Jeong, S.-S.; Gready, J. E. Anal. Biochem. 1994, 221, 273-277.
(17) You, K.-s.; Arnold, L. J.; Allison, W. S.; Kaplan, N. O. TIBS 1978,
3, 265-268.
(15) Mostad, S. B.; Helming, H. L.; Groom, C.; Glasfeld, A. Biochem.
Biophys. Res. Commun. 1997, 233, 681-686.
Org. Lett., Vol. 1, No. 6, 1999
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