Stereospecific deuteration of 2-deoxyerythrose 4-phosphate using 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

Article abstract of DOI:10.1016/j.bmcl.2005.02.080

Racemic 2-deoxyerythrose 4-phosphate was synthesized and one enantiomer of this compound was found to be a substrate for Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. When the reaction was carried out in deuterium oxide, an enzyme-catalyzed regio- and stereoselective incorporation of deuterium into the product was observed.

Full text of DOI:10.1016/j.bmcl.2005.02.080

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2339–2342
Stereospecific deuteration of 2-deoxyerythrose 4-phosphate using
3-deoxy-D-arabino-heptulosonate 7-phosphate synthase
Rachel M. Williamson, Amy L. Pietersma, Geoffrey B. Jameson and Emily J. Parker*
Institute of Fundamental Sciences, Massey University, Palmerston North 5301, New Zealand
Received 21January 2005; revised 24 February 2005; accepted 28 February 2005
Available online 25 March 2005
Abstract—Racemic 2-deoxyerythrose 4-phosphate was synthesized and one enantiomer of this compound was found to be a sub-
strate for Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. When
the reaction was carried out in deuterium oxide, an enzyme-catalyzed regio- and stereoselective incorporation of deuterium into the
product was observed.
Ó 2005 Elsevier Ltd. All rights reserved.
The enzyme 3-deoxy-D-arabino-heptulosonate 7-phos-
phate (DAH7P) synthase catalyzes the first committed
step of the shikimate pathway. This biosynthetic path-
way occurs in plants and microorganisms, but not in
mammals, and is responsible for the formation of aro-
matic compounds including the aromatic amino acids.1
As the biosynthesis of aromatic compounds is required
as a part of primary metabolism in many pathogenic
organisms, the enzymes of the shikimate pathway are
potential targets for new antimicrobial compounds.2–5
charomyces cerevisiae.6–11 These studies have shown
that the reaction involves an unusual cleavage of the
C–O bond in PEP, and is likely to proceed through a
linear bisphosphate intermediate formed by a si-face at-
tack of the C3 of PEP on the C1 re-face of E4P.12,13
However, several aspects regarding the interaction of
the enzyme with its substrates and their subsequent acti-
vation for catalysis remain unresolved. DAH7P syn-
thases belong to a family of enzymes that also
includes the KDO8P synthases.14,15 These enzymes
catalyze an analogous reaction between PEP and a
five-carbon phosphorylated monosaccharide, arabinose
5-phosphate.
DAH7P synthase catalyzes a stereospecific aldol-like
condensation between phosphoenolpyruvate (PEP) 1
and erythrose 4-phosphate (E4P) 2 to give DAH7P 3
and inorganic phosphate (Fig. 1). Many of the features
of this enzymic reaction have been revealed through
labelling studies and through structural analyses of
the DAH7P synthases from Escherichia coli and Sac-
While there have been previous reports detailing the
interaction of five-carbon phosphorylated sugars with
DAH7P synthase,16,17 there has been no investigation
of the role(s) that the erythrose 4-phosphate hydroxyl
groups play in binding and catalysis. In contrast, 2-, 3-,
and 4-deoxyarabinose 5-phosphate derivatives have been
tested on KDO8P synthases, and it has been shown that
the 2- and 3-hydroxyl groups are important in this
enzyme-catalyzed reaction.18,19 We report here the syn-
thesis of racemic 2-deoxyerythrose 4-phosphate (2-
deoxyE4P), its interaction with the phenylalanine-sensi-
tive DAH7P synthase from E. coli, and the isolation of
3,5-dideoxy-D-arabino-heptulosonate 7-phosphate. This
is the first report of the interaction of an alternative
four-carbon aldose phosphate substrate with this key
biosynthetic enzyme.
Figure 1. Reaction catalyzed by DAH7P synthase.
Keywords: Shikimate; Erythrose 4-phosphate; 3-Deoxy-D-arabino-
heptulosonate 7-phosphate; Deoxyerythrose 4-phosphate.
*
2-DeoxyE4P was prepared in a four-step synthesis com-
mencing with allyl chloride 4, following a procedure
Corresponding author. Tel.: +64 356 9099x3566; fax: +64 6 350
5682; e-mail: e.j.parker@massey.ac.nz
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.080

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R. M. Williamson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2339–2342
Scheme 1. Chemical synthesis of racemic 2-deoxyE4P 8 followed by enzymic preparation of 3,5-dideoxy-D-arabino-heptulosonate 7-phosphate 9. (i)
Mg, CH(OEt)3, 37%. (ii) mCPBA, CH2Cl2, 94%. (iii) KH2PO4, reflux, 68%. (iv) (1) Dowex-H+, (2) Dowex-Na+, 79% (v) DAH7P synthase, Mn2+
,
H2O, pH 6.8, 50%. (vi) DAH7P synthase, Mn2+, D2O, pD 6.8, 50%.
20
´
similar to that outlined by Andre et al. and Guerard
et al.21 (Scheme 1). Allyl chloride was converted into
4,4-diethoxy-1-butene 5 by formation of the Grignard
derivative and treatment with triethyl orthoformate.22
Epoxidation with meta-chloroperbenzoic acid in
dichloromethane gave 4,4-diethoxy-1,2-epoxybutane 6.
The epoxide ring was then opened using inorganic phos-
phate to give 4,4-diethoxy-2-hydroxybutyl phosphate,
which was then isolated as its disodium salt 7. The alde-
hyde moiety was released using Dowex-H+ cation
exchange resin. Treatment with Dowex-Na+ and sub-
sequent lyophilization of the aqueous solution gave
2-deoxyE4P 8 as its disodium salt.
The reaction catalyzed by DAH7P synthase is routinely
monitored by the loss of PEP at 232 nm in the presence
of E4P.23 2-DeoxyE4P was substituted for E4P in the
enzyme assay and a time-dependent decline in absor-
bance was recorded, at a rate proportional to the
DAH7P synthase concentration. The total change in
absorbance (with PEP in excess) was proportional to
the amount of 2-deoxyE4P added, but corresponded
to the consumption of only 50% of the racemic 2-deoxy-
E4P present in the solution, suggesting that only a single
enantiomer was acting as a substrate. The steady state
kinetic parameters for this reaction were determined
giving a KM value of 650 lM for 2-deoxyE4P and a kcat
of 14 sÀ1. While this corresponds to a specificity
constant more than two orders of magnitude lower than
that determined for the natural substrate (KM(E4P)
=
21 lM, kcat(E4P) = 71 sÀ1), 2-deoxyE4P is the best alter-
native substrate for E. coli DAH7P synthase so far
reported.
To confirm this specificity, and to identify the product of
the enzymic reaction, the product was prepared on a lar-
ger scale. 2-DeoxyE4P (4.5 mg) and PEP (4.5 mg) were
dissolved in water (0.5 mL), manganese sulfate was
added (to a final concentration of 60 lM), and the pH
was adjusted to 6.8 using 10% sodium hydroxide solu-
tion. The enzymic reaction was then initiated by the
addition of E. coli DAH7P synthase (20 lg). This
Figure 2. 1H NMR spectra (400 MHz) of (a) DAH7P (b) 5-deoxy-
DAH7P (c) (5S)-[5-2H]-3,5-dideoxy-D-arabino-heptulosonate 7-phos-
phate. Minor peaks are attributed to the presence of small
concentrations of the b-anomer.

R. M. Williamson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2339–2342
2341
reaction was monitored by the loss of PEP absorbance
at 260 nm.24 When the loss of PEP had ceased, the en-
zyme was removed by ultrafiltration and the solution
was applied to an anion exchange column.25 Fractions
containing product were pooled, lyophilized, and ana-
or when the nondeuterated 5-deoxyDAH7P is incubated
with enzyme and phosphate in D2O. These observations,
combined with the stereospecificity of the deuterium
incorporation and the predicted relative acidity of the
C2 protons in 2-deoxyE4P, indicate that the deuteration
occurs when (3S)-2-deoxyE4P is bound to the enzyme
prior to reaction with PEP. As the deuterium is observed
in the 5S position only (the position corresponding
to the C2 hydroxyl group in E4P), it is tempting to
speculate that the exchange is catalyzed by an enzymic
residue normally responsible for forming a hydrogen
bond contact to the C2 hydroxyl group of the natural
substrate.
1
lyzed by H NMR spectroscopy (Fig. 2b). Comparison
of this spectrum with that obtained when DAH7P 3
was prepared and purified using the same method
(Fig. 2a) indicated that 3,5-dideoxy-D-arabino-heptulo-
sonate 7-phosphate (5-deoxyDAH7P) 9 had been iso-
lated. This product arises from the reaction of (3S)-2-
deoxyE4P with PEP catalyzed by DAH7P synthase.26
This enzyme therefore shows preferential binding and
catalysis of the 2-deoxyE4P enantiomer with the same
C3 configuration as the natural substrate E4P. As this
product is isolated with the arabino configuration, this
enzyme-catalyzed reaction proceeds with the same facial
selectivity with respect to its monosaccharide substrate
as is observed with E4P. No product with the alternative
stereochemistry at C6 arising from reaction between
PEP and the other enantiomer, (3R)-2-deoxyE4P, was
observed.
While to date there is no structure that shows E4P
bound in the active site of DAH7P synthase in a manner
competent for reaction, E4P has been modelled into the
enzyme active site based on the binding of the nonreac-
tive E4P analogue glycerol 3-phosphate (Fig. 3).10 This
model suggests that the C2 hydroxyl group of E4P is
orthogonal to the carbonyl bond and interacts with
Arg165 (proton donor) and the main chain carbonyl
of Pro98 (proton acceptor), and that activation of the
E4P carbonyl by metal coordination initiates the enzy-
mic reaction. Coordination of (3S)-2-deoxyE4P in a
similar manner would be expected to significantly
increase the acidity of the proR proton on C2 allowing
for the stereospecific deuteration observed in these
studies.
In order to confirm the use of a single enantiomer of 2-
deoxyE4P, the enzyme-catalyzed reaction was moni-
tored by H NMR in deuterium oxide. In this experi-
1
ment, formation of a single product was observed, but
signals corresponding to three methylene protons only
were observed in the product spectrum (Fig. 2c). This
product was isolated by anion exchange chromatogra-
phy, and identified as (5S)-[5-2H]-3,5-dideoxy-D-arabino-
heptulosonate 7-phosphate 10, using mass spectral and
2D NMR analysis. Interestingly, the deuterium is
observed only in the 5S position. Under identical condi-
tions without DAH7P synthase present, no exchange of
deuterium at C2 of 2-deoxyE4P was observed. No incor-
poration of deuterium into DAH7P is found when the
enzymic reaction is carried out with natural substrates,
In summary, we report a short synthesis of racemic 2-
deoxyE4P and the reaction of a single enantiomer of this
compound with DAH7P synthase. Enzymic reaction led
to the isolation of 5-deoxyDAH7P 9, a previously unre-
ported compound. When the reaction is carried out in
deuterium oxide a novel stereospecific enzyme-catalyzed
deuteration of the product at C5 is observed. Detailed
kinetic analyses of the interaction of these compounds
Figure 3. Model of E4P and (3S)-2-deoxyE4P in the active site of E. coli DAH7P synthase. The region of interest for the stereo- and regiospecific
H/D exchange is highlighted. Model based on PEP and Mn2+ containing enzyme8 (1n8f) and the S. cerevisiae structure10 (1of8) containing Co2+, PEP
and glycerol 3-phosphate. Hydrogen bonds are shown as dotted lines and key interactions as dashed lines. Residues K97 and T100 are omitted, and
only selected hydrogens in E4P, 2-deoxyE4P and R165 are shown for clarity.

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R. M. Williamson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2339–2342
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These studies were funded by the Royal Society of New
Zealand Marsden Fund (MAU008).
15. Raetz, C. R. H.; Whitfield, C. Annu. Rev. Biochem. 2002,
71, 635.
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24. A higher wavelength than the kmax for PEP was used to
keep absorbances within the linear range of the
spectrophotometer.
25. Amersham SourceQÒ (8 mL bed volume) eluted with a
linear gradient of 0–500 mM ammonium bicarbonate.
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deoxyerythrose 4-phosphate have discounted the unlikely
possibility that this product arises from reaction of PEP
on the si-face of (3R)-2-deoxyE4P.