Investigating the role of the hydroxyl groups of substrate erythrose 4-phosphate in the reaction catalysed by the first enzyme of the shikimate pathway

Article abstract of DOI:10.1016/j.bmcl.2011.09.017

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P) synthase catalyses the first step of the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids in microorganisms and plants. This enzyme catalyses an aldol reaction between phosphoenolpyruvate and d-erythrose 4-phosphate to generate DAH7P. Both 2-deoxyerythrose 4-phosphate and 3-deoxyerythrose 4-phosphate were synthesised and tested as alternative substrates for the enzyme. Both compounds were found to be substrates for the DAH7P synthases from Escherichia coli, Pyrococcus furiosus and Mycobacterium tuberculosis, consistent with an acyclic mechanism for the enzyme for which neither C2 nor C3 hydroxyl groups are required for catalysis. The enzymes all showed greater tolerance for the loss of the C2 hydroxyl group than the C3 hydroxyl group.

Full text of DOI:10.1016/j.bmcl.2011.09.017

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6838–6841
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Investigating the role of the hydroxyl groups of substrate erythrose
4-phosphate in the reaction catalysed by the first enzyme
of the shikimate pathway
David Tran a, Amy L. Pietersma b, Linley R. Schofield b,c, Matthias Rost b, Geoffrey B. Jameson b,
Emily J. Parker a,
⇑
a Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand
b Institute of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand
c AgResearch Ltd, Grasslands Research Centre, Tennent Drive, Private Bag 11 008, Palmerston North 4442, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Deoxy-
pathway, which is responsible for the biosynthesis of aromatic amino acids in microorganisms and plants.
This enzyme catalyses an aldol reaction between phosphoenolpyruvate and -erythrose 4-phosphate to
generate DAH7P. Both 2-deoxyerythrose 4-phosphate and 3-deoxyerythrose 4-phosphate were synthes-
ised and tested as alternative substrates for the enzyme. Both compounds were found to be substrates
for the DAH7P synthases from Escherichia coli, Pyrococcus furiosus and Mycobacterium tuberculosis, consis-
tent with an acyclic mechanism for the enzyme for which neither C2 nor C3 hydroxyl groups are required
for catalysis. The enzymes all showed greater tolerance for the loss of the C2 hydroxyl group than the C3
hydroxyl group.
D-arabino-heptulosonate 7-phosphate (DAH7P) synthase catalyses the first step of the shikimate
Received 8 August 2011
Revised 4 September 2011
Accepted 6 September 2011
Available online 16 September 2011
D
Keywords:
DAH7PS
Substrate specificity
Erythrose 4-phosphate
Aromatic amino acid biosynthesis
Shikimate pathway
Ó 2011 Elsevier Ltd. All rights reserved.
3-Deoxy-
thase catalyses the stereospecific aldol-like condensation reaction
between phosphoenolpyruvate (PEP, 1) and -erythrose 4-phos-
D
-arabino-heptulosonate phosphate (DAH7P, 3) syn-
tuberculosis, Helicobacter pylori and Pseudomonas aeruginosa. All
types and subtypes share a catalytic (b/ barrel, although there
are quite distinct differences in quaternary structure and extra-bar-
rel extensions associated with different mechanisms of allosteric
inhibition of the enzyme.6–10
a
)
8
D
phate (E4P, 2) to generate DAH7P (Fig. 1). This is the first enzyme-
catalysedreactionof theshikimatepathway. Theshikimatepathway
is responsible for biosynthesis of the aromatic amino acids phenylal-
anine, tyrosine and tryptophanas wella range of importantaromatic
metabolites.1 The pathway is present in plants and microorganisms
yet absent in higher organisms. Thus, as the biosynthesis of aromatic
compounds is required for a number of significant pathogens DAH7P
synthase has been identified as a target for antimicrobial drug
design.2,3
Two distinct types of DAH7P synthases have been reported and
although these types share very little sequence identity there is
close correspondence of their structures and active site architec-
ture.4,5 Type I enzymes are found in both bacteria and fungi. The
type I enzymes can be further divided into two groups on the basis
Many key features of the reaction mechanism catalysed by
DAH7P synthase have been elucidated.7–9,11,12 It is known that
the reaction proceeds with cleavage of the CAO bond of PEP. Label-
ling experiments have clearly shown that the si-face of PEP attacks
the re-face of E4P to generate the new CAC bond of product DAH7P
stereospecifically.11–13 All DAH7P synthases require a divalent me-
tal ion for catalytic activity. It is likely that this metal ion is respon-
sible for coordinating the carbonyl functionality of E4P and the
reaction is thought to be initiated by attack of C3 of PEP on the acti-
vated C1 of E4P (Fig. 2). The reaction can potentially take two
alternative pathways following this CAC bond formation. The car-
bonium intermediate could be attacked by water to give a linear
tetrahedral intermediate, which, following elimination of phos-
phate, is released from the enzyme as the acyclic form of DAH7P.
Alternatively, the cation could be captured by the C3 hydroxyl
group of E4P giving a cyclic tetrahedral intermediate and yielding
of sequence, denoted types I
a and Ib; these two groups share
approximately 30% identity. The type II enzymes were originally
found in plants but are now known to be present in a number of
bacteria including significant human pathogens Mycobacterium
the cyclic pyranose form of DAH7P directly. The
a-pyranose form
of DAH7P is the most predominant form of this compound in solu-
tion.14 Current evidence favours the formation of the linear inter-
mediate; this is based on the structure of the active site, which
⇑
Corresponding author. Tel.: +64 3 364 2871; fax: +64 3 364 2110.
E-mail address: Emily.parker@canterbury.ac.nz (E.J. Parker).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.017

D. Tran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6838–6841
6839
O
OH
O
OH
MeOH, SOCl2,
reflux
OH
OMe
MeO
HO
100%
5
O
O
4
BnBr, Ag2O
CH2Cl2
75%
LiAlH4,THF
reflux
O
OBn
OBn
OMe
OH
63%
MeO
HO
O
7
6
8: 18%
9: 6%
10: 6%
Cl-P(O)(OPh)2,
Im, CH2Cl2
OBn
O
OBn
DMP, CH2Cl2
85%
Figure 1. Reaction catalysed by DAH7P synthase.
H
OR1
(PhO)2PO
R2O
O
11
8: R1=H, R2=P(O)(OPh)2
9: R1=P(O)(OPh)2, R2=H
10: R1=R2=P(O)(OPh)2
would seem to support a linear intermediate, and on recent inhibi-
tion studies.7,8,15 However, the role of a cyclic intermediate, even as
part of product release or phosphate elimination has not been ruled
out.
Our aim in these studies was to examine the roles that both the
2- and 3-hydroxyl groups play in both substrate binding and catal-
ysis for each of the different types of DAH7P synthase. In order to
do this, 2- and 3-deoxyE4P substrate analogues were synthesized
and tested as alternative substrates for DAH7P synthases from dif-
ferent organisms.
CH(OMe)3,
H2SO4,
MeOH
94%
1. PtO2, H2
MeOH
OBn
OH
O
OMe
2: H2O, 40oC
H
(PhO)2PO
H2O3PO
OMe
O
13
12
Scheme 1. Synthesis of 3-deoxyE4P.
The synthesis of 2-deoxyE4P in both enantiopure and racemic
forms has been previously described.16,17 For the synthesis of
3-deoxy E4P (Scheme 1), (R)-malic acid 4 was first esterified to give
its dimethyl ester 5. The secondary hydroxy group was then pro-
tected as the benzyl ether, and the ester functionalities were re-
duced using LiAlH4 to give diol 7. Phosphorylation of this diol
produced the isomeric monophosphorylated products 8 and 9
and the diphosphorylated compound 10 in a 3:1:1 ratio. A signifi-
cant amount of unreacted starting material was also recovered
(24%). The best yields of the desired monophosphorylated product
8 were obtained using 0.9 equiv of the phosphorylating reagent.
Oxidation of phosphate 8 using Dess–Martin periodinane (DMP)
gave aldehyde 11. The aldehyde functionality was protected as
its dimethyl acetal prior to hydrogenolysis. Final deprotection gave
(R)-3-deoxyE4P 13. Racemic (RS)-3-deoxyE4P was also synthesised
using the same route starting with DL-malic acid.
Both 2-deoxyE4P and 3-deoxyE4P were tested as alternative
substrates for DAH7P synthases representing each of the three dif-
ferent types and subtypes. Specifically, DAH7P synthases from Esch-
erichia coli (type Ia), the hyperthermophilic archeon Pyrococcus
furiosus (type Ib), and the organism responsible for tuberculosis,
M. tuberculosis (type II) were employed. All these enzymes have
been both structurally and functionally characterised.9,18–20 Both
2- and 3-deoxyE4P analogues were substrates for all three enzymes
(Table 1).
Apart from (S)-2-deoxyE4P, which intriguingly was a consider-
ably better substrate for the P. furiosus enzyme than the natural
substrate E4P, the deoxyE4P analogues were generally found to
be poorer substrates than E4P. Catalysis by M. tuberculosis DAH7P
synthase was only marginally impaired when 2-deoxyE4P was
used, but the rate of reaction catalysed by the E. coli enzyme was
quite significantly impaired, substantially due to a significantly
higher KM value for this alternative substrate.
(R)-3-DeoxyE4P was a substrate for all the enzymes, ruling out
an essential role for the 3-hydroxyl group in the mechanism of this
enzyme, and an obligate cyclic mechanism for the DAH7P synthase
(Fig. 2). This result is entirely consistent with the active-site
Figure 2. Acyclic (left) and cyclic (right) mechanisms for the formation of DAH7P.

6840
D. Tran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6838–6841
Table 1
Kinetic parameters of E4P, 2-deoxy and 3-deoxyE4P with DAH7P synthases from different organisms, representing each of the different types and subtypes of DAH7P synthase
E4P
(S)-2-DeoxyE4P
(R)-3-DeoxyE4P
DAH7P synthase source (and type)a
E. coli (type I
a
)
KM
kcat (sÀ1
cat=KEM4P (sÀ1
(
l
M)
)
39
26
0.67
4
2
410 40
2700 140
4.5 0.1
0.002
25
3
k
l
l
l
MÀ1
MÀ1
MÀ1
)
)
)
0.06
P. furiosus (type Ib)
KM M)
kcat (sÀ1
cat=KEM4P (sÀ1
KM M)
kcat (sÀ1
cat=KEM4P (sÀ1
(l
9
1
6
1
200 30
2.1 0.1
0.011
)
1.4 0.1
0.16
3.0 0.1
0.5
k
M. tuberculosis (type II)
(l
37
5.4 0.1
0.15
2
46
5
77 10
2.4 0.1
0.031
)
4.9 0.2
0.11
k
a DAH7P synthases from all sources were assayed in accordance with previously reported procedures as described in the Supplementary data the presence of Mn2+. The E. coli
enzyme used in these studies is the phenylalanine-sensitive isozyme.21
architecture of the enzyme and very recent studies indicating that
a linear tetrahedral intermediate is an efficient inhibitor of the M.
tuberculosis enzyme.15
Whereas (R)-3-deoxyE4P was a substrate for all three enzymes,
this compound was found to be a substantially poorer substrate
the C2 hydroxyl group and the main-chain carbonyl oxygen of
the conserved proline in the E4P binding pocket, whereas in the
P. furiosus DAH7PS structure this proline (in the absence of E4P)
is observed to be disordered. The absence of this interaction may
account for the observation that (S)-2-deoxyE4P is a relatively poor
substrate for the E. coli enzyme.
The racemic forms of the deoxy substrate analogues were
also tested on the enzymes. Both the E. coli and M. tuberculosis
enzymes utilised only half of the total concentration of the racemic
2-deoxyE4P mixture, indicating that only (3S)-2-deoxyE4P, with
the correct C3 stereochemistry, was indeed a substrate for these
enzymes. In marked contrast, the P. furiosus enzyme was far more
promiscuous, with both enantiomers of 2-deoxyE4P acting as
alternative substrates. However, the rates of reaction were not
identical, and clear kinetic resolution of the mixture was observed
through reaction with the enzyme, as indicated by the initial fast
use of the (3S)-2-deoxyE4P enantiomer, followed by significantly
slower consumption of (3R)-2-deoxyE4P (Fig. 4).
In contrast to the promiscuity of the P. furiosus DAH7P synthase
towards 2-deoxyE4P substrates, racemic 3-deoxyE4P was not a sub-
strate for the enzyme, consistent with significant inhibition of this
enzyme and nonproductive binding with (S)-3-deoxyE4P prevent-
ing productive binding by (R)-3-deoxyE4P. This result was unex-
pected given the tolerance of this enzyme to the loss of the C2
hydroxyl group. Even more surprisingly, we observed that E. coli
DAH7P synthase was able to tolerate variation in the configuration
at C3 with the racemic 3-deoxyE4P substrate. Indeed, 100% utilisa-
tion of the racemic mixture was observed and the KM determined
than both (S)-2-deoxyE4P and
larly apparent for the type I
D
-E4P in all cases. This was particu-
a
E. coli DAH7P synthase, for which
(R)-3-deoxyE4P is a very poor substrate exhibiting a high Michaelis
constant and a specificity constant over 300 times smaller than
that determined for the natural substrate, E4P. On the other hand
for M. tuberculosis DAH7P synthase, a corresponding diminution
in specificity constant of only five-fold was observed.
Modelling of E4P into the active site of DAH7P synthase pro-
vides some insight into why the C3 hydroxyl of E4P is more impor-
tant to substrate binding and catalysis than the C2 hydroxyl group
(Fig. 3). The essential divalent metal found in the active site of
DAH7P synthase is coordinated by four residues, Cys, Glu, His
and Asp. These residues are absolutely conserved in all DAH7P syn-
thases types. Modelling of E4P into the active site of the enzyme
indicates that the metal-coordinating Asp residue is also predicted
to hydrogen bond to the C3 hydroxyl. Removal therefore of this hy-
droxyl group not only removes a key binding contact of the aldehy-
dic substrate but also would be expected to alter the reactivity of
the metal centre, and hence the activation of the carbonyl group.
In P. furiosus and E. coli DAH7PS the C2 hydroxyl appears to
make a hydrogen bond to an active-site Arg in the PEP binding site
(Arg115 P. furiosus DAH7PS numbering). In the E. coli DAH7P
synthase there is also a likely hydrogen-bonding contact between
Figure 3. Active site of DAH7P synthase with E4P modelled into position so as to interact with the metal ion and expose the re-face of the aldehyde moiety to PEP. (A) E. coli
DAH7P synthase, (B) P. furiosus DAH7P synthase and (C) M. tuberculosis DAH7P synthase.

D. Tran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6838–6841
6841
Figure 4. Reaction of racemic (RS)-2-deoxyE4P catalysed by P. furiosus DAH7P synthase, showing initial fast rate corresponding to the use of (3S)-2-deoxyE4P (equivalent to
the rate of reaction in the presence of the enantiopure (3S)-2-deoxyE4P) followed by slow reaction rate due to consumption of the (3R)-2-deoxyE4P. The reaction progress is
followed by the loss of PEP at 232 nm as described in the Supplementary data.
for this mixture was virtually unchanged (2600 330
l
M)
article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.09.017.
– although, it should be noted that KM for (S)-2-deoxyE4P and (R)-
3-deoxyE4P are very much larger compared to E4P for the E. coli
DAH7P synthase than for the M. tuberculosis and P. furiosus DAH7P
synthases. In contrast the type II M. tuberculosis DAH7P synthase
was intolerant to variation of configuration at C3, with the racemic
3-deoxyE4P substrate utilising only 50% of the racemic mixture,
presumably the (R)-3-deoxyE4P based on the enantiopure results.
The conformational flexibility that allows both enantiomers of
2-deoxyE4P to be substrates for P. furiosus DAH7PS allows the (S)
enantiomer of 3-deoxyE4P to bind in a manner that precludes the
binding and reaction observed for enantiopure (R)-3-deoxyE4P.
In summary, both 2- and 3-deoxy variants of E4P were found to
be substrates for both types I and II DAH7P synthases. This indi-
cates that whereas the hydroxyl groups, and in particular the C3
hydroxyl group of this aldehydic substrate support efficient reac-
tion, they are not essential for DAH7P synthase catalysis. The
observation that 3-deoxyE4P acts as an alternative substrate
strongly supports an acyclic reaction mechanism for the genera-
tion of DAH7P by this enzyme.
References and notes
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We gratefully acknowledge the New Zealand Marsden Fund
(UOC710) and the Massey University Research Fund for financial
support for this project, and the Department of Chemistry at the
University of Canterbury for financial assistance to D.T.
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Supplementary data
Supplementary data associated (details of synthetic procedures
for the preparation of 3-deoxyE4P and the kinetic assays) with this