Arabinose 5-phosphate analogues as mechanistic probes for Neisseria meningitidis 3-deoxy-d-manno-octulosonate 8-phosphate synthase

Article abstract of DOI:10.1016/j.bmc.2008.09.056

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the condensation reaction between phosphoenolpyruvate and d-arabinose 5-phosphate (d-A5P) in a key step in lipopolysaccharide biosynthesis in Gram-negative bacteria. The KDO8P synthase from Neisseria meningitidis was cloned into Escherichia coli, overexpressed and purified. A variety of d-A5P stereoisomers were tested as substrates, of these only d-A5P and l-X5P were substrates. The Asn59Ala mutant of N. meningitidis KDO8P synthase was constructed and this mutant retained less than 1% of the wild-type activity. These results are consistent with a catalytic mechanism for this enzyme in which the C2 and C3 hydroxyl groups of d-A5P and Asn59 are critical.

Full text of DOI:10.1016/j.bmc.2008.09.056

Bioorganic & Medicinal Chemistry 16 (2008) 9830–9836
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Arabinose 5-phosphate analogues as mechanistic probes for Neisseria
meningitidis 3-deoxy-D-manno-octulosonate 8-phosphate synthase
Meekyung Ahn a, Fiona C. Cochrane a, Mark L. Patchett b, Emily J. Parker c,
*
a Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
b Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand
c Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 June 2008
Revised 21 September 2008
Accepted 23 September 2008
Available online 26 September 2008
3-Deoxy-
between phosphoenolpyruvate and
biosynthesis in Gram-negative bacteria. The KDO8P synthase from Neisseria meningitidis was cloned into
Escherichia coli, overexpressed and purified. A variety of -A5P stereoisomers were tested as substrates, of
these only -A5P and -X5P were substrates. The Asn59Ala mutant of N. meningitidis KDO8P synthase was
constructed and this mutant retained less than 1% of the wild-type activity. These results are consistent
D
-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the condensation reaction
D
-arabinose 5-phosphate ( -A5P) in a key step in lipopolysaccharide
D
D
D
L
Keywords:
Neisseria meningitidis
with a catalytic mechanism for this enzyme in which the C2 and C3 hydroxyl groups of
are critical.
D-A5P and Asn59
D-A5P
Ó 2008 Elsevier Ltd. All rights reserved.
KDOP
Lipopolysaccharide biosynthesis
2—
1. Introduction
OPO
3
OH
—
2—
O C
OPO
3
2
HO
HO
KDO8P synthase
3-Deoxy-
synthase (EC 2.5.1.55) catalyses the condensation reaction
between phosphoenolpyruvate (PEP) and -arabinose 5-phosphate
-A5P) to generate KDO8P and inorganic phosphate (see Fig. 1).
This reaction is the first committed step in the biosynthesis of 3-
deoxy- -manno-octulosonate (KDO), a site-specific component of
D-manno-octulosonate 8-phosphate synthase (KDO8P)
O
—
CO
2
PEP
O PO
D
OH
O
P
i
OH
KDO8P
(D
2—
H
3
OH OH
D-A5P
D
the inner core of the lipopolysaccharide (LPS), and is essential for
LPS formation.1 As the LPS is found in most Gram-negative bacteria
and is necessary for their viability, the enzymes responsible,
including KDO8P synthase, have been identified as attractive tar-
gets for the development of novel antimicrobial compounds.
The reaction mechanism of KDO8P synthase has already
received considerable attention. It has been shown that the con-
densation follows an ordered sequential kinetic mechanism in
Figure 1. Reaction catalysed by KDO8P synthase.
theless, the precise catalytic events that lead to this intermediate
are still unresolved. These initial studies were performed primarily
with the KDO8P synthase from Escherichia coli, an enzyme, that is,
unaffected by the presence of metal ions or metal ion chelators.
However, more recently it has been shown that the KDO8P syn-
thases from Aquifex aeolicus6, Helicobacter pylori7, Chlamydia psit-
taci7 and Aquifex pyrophilus8 require the presence of a metal ion
for activity, raising the possibility that two distinct families of
KDO8P synthases with different catalytic mechanisms exist.
Structures of both metal-dependent (A. aeolicus) and metal-
independent (E. coli) KDO8P synthases have been determined.9,10
which PEP binds to the enzyme before
D-A5P and the product
KDO8P is released last.2 Labeling studies have shown that the reac-
tion is highly stereospecific with the si face of PEP coupling with
the re face of D-A5P, and that the reaction involves the unusual
cleavage of the C–O bond of PEP rather than O–P bond cleavage.3,4
A linear biphosphate reaction intermediate arising from attack of
water at C2 of PEP following attack by C3 of PEP on the electro-
philic carbonyl carbon of acyclic D-A5P has been identified using
Both enzymes crystallise as homotetramers with (b/a8-barrel sub-
time-resolved electrospray ionisation mass spectrometry.5 Never-
units containing four independent active sites. Almost all key
active site residues appear to be conserved in both proteins with
the only notable difference being at the metal-binding site.
Three of the four residues responsible for metal coordination in
* Corresponding author. Tel.: +64 3 364 2100; fax: +64 3 364 2110.
E-mail address: Emily.Parker@canterbury.ac.nz (E.J. Parker).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.056

M. Ahn et al. / Bioorg. Med. Chem. 16 (2008) 9830–9836
9831
Table 1
A. aeolicus KDO8P synthase are found in the metal-independent
Purification of N. meningitidis wild-type KDO8P synthase
E. coli KDO8P synthase. The fourth ligand, a Cys, is substituted by
an Asn in the metal-independent E. coli enzyme.7 Sequence analy-
sis indicates that all KDO8P synthases whose activity has been
shown to be metal-dependent have this Cys, whereas the Asn is
found in the equivalent position in characterised metal-indepen-
dent enzymes, indicating that this amino-acid may be the prime
determinant of metal dependency. Recent elegant studies have
shown that single reciprocal mutations between the Cys and Asn
interconvert metal-dependent and metal-independent enzyme
activity, although with heavily compromised activity relative to
the parent enzyme.11 As the ability to bind metal is readily dis-
pensable it is likely that both metal-independent and metal-
dependent enzymes share a common reaction mechanism, and
that the metal (when present) plays a structural rather than cata-
lytic role.
Step
Total
protein
(mg)
Total
enzyme
(U)
Calculated
specific
Yield
(%)
Approximate
purity
activity
(U mgꢁ1
0.6
)
a
Crude cell
lysate
240
140
100
1.00
TM
Source 15Q
Source 15Phe
66
48
115
96
1.7
2.0
28
20
3.0
3.5
TM
and Concentration
a
Specific activity was determined at 25 °C and pH 7.5.
and a single band of ꢀ 30,000 Da was visible by SDS–PAGE. The
NmeN59A mutant was also overexpressed and again found to be
predominantly soluble. Once purified the activity of the mutant
was determined and was found to have only 0.5% of the wild-type
KDO8P synthase is closely related to the enzyme that catalyses
activity for substrate D-A5P.
the first step of the shikimate pathway, 3-deoxy-
sonate 7-phosphate (DAH7P) synthase; DAH7P synthase catalyses
the formation of DAH7P from PEP and the four-carbon sugar -ery-
throse 4-phosphate ( -E4P) in an analogous reaction. DAH7P and
D-arabino-heptulo-
Consistent with the sequence analysis suggesting that this
enzyme has an asparagine residue (Asn23) rather than the metal-
binding cysteine, the enzyme does not appear to require a metal
for catalysis. Assays enriched with Mn2+ did not alter activity,
and enzyme activity was unchanged by treatment with 1 mM
EDTA (data not shown).
D
D
KDO8P synthases share many mechanistic and structural features
and on the basis of this similarity and detailed phylogenetic analyses
it has been proposed that these enzymes share a common ancestry.7
Whereas DAH7P synthase appears to be relatively insensitive to
changes in its aldehydic substrate KDO8P synthases are reported
to exhibit greater substrate stringency.12–14 Previous studies have
2.2. Preparation of D-A5P substrate analogues
A series of A5P analogues was prepared by a combination of
chemical and enzymatic syntheses in order to investigate the influ-
ence of hydroxyl group configuration on the KDO8P synthase cat-
shown that the C2 epimer of
-R5P),15,16 is not a substrate for either metal-dependent or me-
tal-independent KDO8P synthases and 2- and 3-deoxy analogues
of -A5P are either very poor substrates or not utilised at all as
A5P alternatives in the KDO8P synthase catalysed reaction.2,12,16,17
D-A5P, ribose 5-phosphate
(D
alysed reaction.
D
-Lyxose 5-phosphate
(D-L5P) was prepared
D
D-
enzymatically from
D
-lyxose using hexokinase in an analogous
reaction to that used for the preparation of D-A5P from D-arabinose
In contrast, acyclic 4-deoxy-D-A5P is reported to be a substrate for
(Fig. 2). 31P NMR spectroscopy was used to follow the progress of
these reactions.
the E. coli KDO8P synthase.2
Neisseria meningitidis, a Gram-negative bacterium that colonises
the nasopharynx of about 10% of healthy humans, is responsible for
two serious human diseases, pyogenic meningitis and meningo-
coccal septicemia.18,19 Sequence alignment of the N. meningitidis
KDO8P synthase with other characterised KDO8P synthases reveals
an Asn residue is present rather than the metal-binding Cys sug-
gesting this enzyme is metal-independent. In this study we de-
scribe the characterisation of the KDO8P synthase from N.
meningitidis and address the specific roles that the C2, C3 and C4
D
-Xylose 5-phosphate (
were synthesised from - and
cedure outlined in Scheme 1.
D
-X5P) and
L-xylose 5-phosphate (L-X5P)
D
L-xylose, respectively, using the pro-
2.3. Substrate specificity and kinetics of N. meningitidis KDO8P
synthase
In order to investigate the substrate specificity and catalytic
mechanism of this enzyme a range of phosphorylated aldopentoses
were tested as substrates and kinetics were determined for those
hydroxyl groups of substrate
reaction. To this end we have prepared the
-lyxose 5-phosphate and - and -xylose 5-phosphate and exam-
ined the ability of these compounds and -R5P to act as substrates
D-A5P play in the KDO8P synthase
D-A5P diastereomers
that showed activity (Table 2). The compounds
D
-R5P,
D-L5P and
D
D
L
D-X5P, with altered configuration at C2, C3 or both C2 and C3 rel-
D
ative to
D
-A5P were not substrates for N. meningitidis KDO8P syn-
-X5P, in which only the C4 position is inverted
relative to natural substrate -A5P, was utilised as a substrate by
this enzyme, albeit to a lesser extent than -A5P as evidenced by
for N. meningitidis KDO8P synthase. Our findings illuminate a cata-
lytic mechanism for KDO8P synthase in which the correct configu-
rations of the C2 and C3 hydroxyl groups, but not the C4 hydroxyl
group, are essential for enzymic reaction.
thase. However,
L
D
D
the 10-fold decrease in the kcat/Km value. This change in enzyme
efficiency resulted from an approximately fivefold increase in the
Km for the phosphorylated monosaccharide. The product of the
2. Results
reaction between PEP and L-X5P was purified and characterised.
2.1. Cloning, purification and properties of N. meningitidis
KDO8P synthase
Consistent with the predicted structure, where the altered stereo-
centre is outside the pyranose or furanose ring, the 1H NMR spec-
trum of this compound, 3-deoxy-L-gulo-octulosonate 8-phosphate,
was very similar to that of the natural product KDO8P (Fig. 3).
The KDO8P synthase from N. meningitidis was successfully
cloned into the pT7-7 plasmid and subsequent SDS–PAGE analysis
determined that the protein was overexpressed in E. coli BL21(DE3)
cells and found predominantly in the soluble fraction. Only two
purification steps were required to yield pure KDO8P synthase.
Substantial separation from contaminants was achieved by anion
exchange chromatography in the first purification step (Table 1).
Hydrophobic interaction chromatography yielded a further in-
crease in KDO8P synthase purity with only a small loss of protein
3. Discussion
3.1. Mechanistic implications of substrate specificity
These results show that in order to act as a substrate for KDO8P
synthase the correct configuration of the C2 and C3 positions is

9832
M. Ahn et al. / Bioorg. Med. Chem. 16 (2008) 9830–9836
PEP
pyruvate
pyruvate kinase
ATP
H
ADP
OH
O
OH O
2—
HO
O PO
H
3
hexokinase
OH OH
OH OH
D-L5P
D-A5P
PEP
PEP
Figure 2. Hexokinase-catalysed preparation of
D-A5P and of
D
-L5P followed by 31P NMR. The reactions were performed at pH 7.6 and at room temperature (ꢀ20 °C).
Table 2
O
O
O
OH
Substrate specificity and kinetic parameters of N. meningitidis KDO8P synthase
i,ii
HO
HO
Substrate structures
kcat
Km
(PEP)
Km
(mono.)
kcat/Km
(sꢁ1
)
(mono.)
HO
OH
HO
O
(lM)
(l
M)
(sꢁ1 Mꢁ1
l )
2
1
D-A5P
2.7 0.6 <1b
12 1f
iii
OH
O
O
O
0.23 0.07
O
2—
O PO
H
H
H
H
3
PhO P O
OPh
OH OH
HO
O
D
-R5Pe
3
OH
O
NSa
2—
iv,v
O PO
3
OH OH
O
HO P O
OH
O
OH
OH
D
-L5Pc
OH
O
HO
NS
2—
O PO
3
5
OH OH
Scheme 1. Synthesis of
D
-xylose 5-phosphate. Reagents and conditions: (i) Me2CO,
D
-X5Pd
H2SO4, CuSO4; (ii) 0.2% HCl; (iii) ClPO(OPh)2, imidazole, CH2Cl2, 31%; (iv) Pt2O, H2,
100%; (v) H2O, 4, 72%.
OH
O
NS
2—
O PO
3
OH OH
crucial, whereas the enzyme is prepared to accept a substrate,
L
-X5P
1.1 0.1 2.5 0.1b 57 2f
L
-X5P with the opposite C4 configuration to that of the natural sub-
strate, -A5P. These results are consistent with the earlier observa-
tions showing that 2-deoxy- -R5P (2-deoxy- -A5P) is at best an
extremely poor substrate for the E. coli enzyme, and that 3-
deoxy-
-A5P is not accepted as a substrate.1,16 The C2 and C3 hy-
droxyl groups of -A5P are clearly required and it is essential that
they be in the correct configuration. Similarly, it has been previ-
ously shown that 4-deoxy- -A5P is a substrate for E. coli KDO8P
synthase2, and this observation in combination with the results
with -X5P presented here suggest that KDO8P synthase is ambiv-
D
OH
O
D
D
0.019 0.003
2—
O PO
3
H
OH OH
D
D
Assays were performed at pH 7.5 and 30 °C.
a
NS - compound was determined not to be a substrate.
b
To determine kinetic parameters for PEP, the assay mixtures contained 1–8
PEP with 80 -A5P or 1–2 M PEP with 300 -X5P.
Assay mixtures contained 160 M PEP and either -L5P (1.5–4 mM),
(0.7–2 mM), or -R5P (1–3 mM; Sigma) with 0.016–0.032 U KDO8P synthase.
To determine kinetic parameters for phosphorylated monosaccharides, the
reaction mixtures contained 160 M PEP with 2–300 -A5P or 7–370 -X5P.
lM
D
l
M
D
l
lM L
c,d,e
l
D
D-X5P
L
D
f
alent to changes at C4.
l
lM
D
lM L
The available structures of A. aeolicus and E. coli KDO8P syn-
thases and the recently proposed mechanism for this enzyme
can be used to rationalise these substrate specificity findings.
Despite the variation in metal dependency, the A. aeolicus and
E. coli enzymes have been shown to share similar active sites,
and these enzymes have, respectively 43% and 68% identity with
the KDO8P synthase from N. meningitidis. The structure of the
A. aeolicus KDO8P synthase bound to both substrates has also
been solved allowing an examination of the interactions that
the hydroxyl groups of the natural substrate -A5P have with
enzyme residues (Fig. 4). The key step in the reaction mecha-
nism catalysed by this enzyme is attack by C3 of PEP on C1 of
D

M. Ahn et al. / Bioorg. Med. Chem. 16 (2008) 9830–9836
9833
Figure 3. 1H NMR spectra (500 MHz) of (a) KDO8P and (b) 3-deoxy-
L
-gulo-octulosonate 8-phosphate (
L-DGO8P), products of the reactions between (a) PEP and
D-A5P, or (b)
PEP and
forms.
L-X5P, respectively, catalysed by N. meningitidis KDO8P synthase. Inserts show an expansion of the signals corresponding to the H3 protons in pyranose and furanose
D
-A5P, and subsequent attack at C2 of PEP by water to form an
gine residue (Asn48 in A. aeolicus, Asn62 in E. coli and Asn59
in N. meningitidis) that also H-bonds to the carbonyl. This residue
is absolutely conserved in all KDO8P synthases sequenced to
date and this interaction would be expected to be critical to
(and have a direct bearing on) the correct positioning of the car-
intermediate, as detected using time-resolved ESI.5 This initial
addition step requires careful placement and activation of the
carbonyl moiety of
C3 of PEP approaches C1 of
tures indicate that the C2 hydroxyl of
water molecule) to the metal in the metal-dependent A. aeolicus
enzyme and an overlay of structures suggests that the C2 will
interact with the absolutely conserved asparagine found instead
of cysteine in the metal-independent KDO8P synthases. This
interaction between C2 hydroxyl and enzyme is important for
controlling the dihedral angle that helps determine positioning
of the carbonyl moiety. Altering the stereochemistry at this posi-
tion would upset this, and consistent with observations made
previously, it would be predicted that altering the configuration
D
-A5P in order to ensure the nucleophilic
-A5P from the correct angle. Struc-
-A5P coordinates (via a
D
D
bonyl moiety of
D-A5P. Hence inversion (this study) or loss of
the C3 hydroxyl group dramatically influences catalysis.1 In line
with this we have found that the NmeN59A mutation of N. men-
ingitidis KDO8P synthase has less than 1% activity of the wild-
type with natural substrate
D-A5P. In contrast, the C4 hydroxyl
group of -A5P makes no obvious interactions that might influ-
D
ence the placement of the carbonyl moiety, and therefore inver-
sion at this centre is not as catastrophic.
4. Conclusion
would have a more significant effect on the ability of D-A5P to
act as a substrate than removing this hydroxyl group completely.
In line with this, the structure of A. aeolicus KDO8P synthase in
In summary, our results with modified substrates and with the
N59A mutant N. meningitidis enzyme are consistent with a mecha-
nism for KDO8P synthase in which the protein interactions with
the C2 and C3 but not the C4 hydroxyl groups are essential for
complex with PEP and
the metal and that there is significant displacement of C1 of
R5P. The structure of the A. aeolicus enzyme with -A5P bound
indicates that the C3 hydroxyl group interacts with an aspara-
D-R5P shows the C2 hydroxyl is closer to
D
-
D
the correct positioning of
PEP’s olefin.
D-A5P’s carbonyl group for attack on
Figure 4. Stereo diagram showing the active site of A. aeolicus KDO8PS (PDB 1FWW9). Metal and metal ligands are in magenta and PEP ligands are shown in slate. Substrates,
PEP and -A5P are shown in green. -A5P ligands are shown in cyan. Asn48 of A. aeolicus KDO8PS, which corresponds to Asn59 of N. meningitidis KDO8PS, is shown in yellow
(note that Asn48 is rotated 180° compared to the published structure for sensible carbonyl ( -A5P)C@O. . .HN(Asn59) hydrogen bond).
D
L
D

9834
M. Ahn et al. / Bioorg. Med. Chem. 16 (2008) 9830–9836
determined to contain mainly the KDO8P synthase were pooled
5. Experimental
and solid (NH4)2SO4 was added (final concentration 1 M). The
TM
protein was loaded onto a Source 15Phe column (Amersham,
5.1. Cloning and expression of N. meningitidis KDO8P synthase
10 ꢂ 105 mm; ambient temperature) pre-equilibrated with Buffer
B (10 mM BTP, pH 7.5, 1 mM DTT, 1 M (NH4)2SO4) at a flow rate
of 1 mL minꢁ1. Nonspecific protein was removed first by washing
the column with Buffer B (3.5 CV) after which a sharp linear gradi-
ent was applied from 1 to 0.68 M (NH4)2SO4 for 0.6 CV. KDO8P syn-
thase was eluted by a linear gradient from 0.68 to 0.3 M (NH4)2SO4
(ꢀ10 CV) during which 3 mL fractions were collected. Protein was
again analysed by SDS–PAGE and fractions that were determined
to be pure KDO8P synthase (or N59A_KDO8P synthase)
were pooled and both concentrated and desalted using a pre-
washed 20 mL Vivaspin concentrator (10,000 MWCO; Vivascience).
An open reading frame, annotated as encoding KDO8P synthase
(kdsA) from N. meningitidis serotype B strain MC58 (ATCC BAA-
355), was retrieved with flanking 50 and 30 regions from Genbank
(http://www.ncbi.nlm.nih.gov/entrez/; Genbank Accession No.
AE002098, gi: 7226523; Protein Accession No. AAF41659). Stan-
dard PCR methodologies were employed to amplify the gene se-
quence from N. meningitidis genomic DNA (ATCC) using the
primers NmeKNdeF (50-GGTGGTCATATGGATATTAAAATCAACGACA
TCAC) and NmeKBamR (50-CGAGGATCCTTTACTCAATTGTTAAAATC
GGTTGTGA) to introduce NdeI and BamHI restriction sites (under-
lined) into the 862 bp PCR product. The synonymous substitution
of A for G in NmeKBamR was introduced to decrease the strong
secondary structure predicted for this oligonucleotide. The ampli-
fied PCR product was recovered directly using a High Pure PCR
product purification kit (Roche), digested with NdeI and BamHI,
ethanol precipitated and ligated into pT7-7,20 which had been pre-
viously digested with the same endonucleases. The resulting con-
struct was used to transform E. coli TG1 (Stratagene) cells. The
sequence of the construct was confirmed using both sense and
antisense strands as templates before the pT7-7-NmekdsA plasmid
was transformed into E. coli BL21(DE3) cells (Novagen).
Site-directed mutagenesis was performed using the Quik-
ChangeÒ II site-directed mutagenesis kit (Stratagene) to create
the Asn59Ala (NmeN59A) mutant of N. meningitidis KDO8P syn-
thase. Mutagenic primers NmeKN59AF 50-CTCTTTCGACAAGGCAGC
ACGTTCCTCCATCCATTC and NmeKN59AR 50-GAATGGATGGAGGAA
CGTGCTGCCTTGTCGAAAGAG (mutation shown in bold) were used
to convert the asparagine to alanine. The sequence of the mutant
was verified, after which the plasmid was transformed into E. coli
BL21(DE3) cells, then the mutant was overexpressed and purified
as described below.
Aliquots (100 lL) of the purified enzymes were flash-frozen
and stored at ꢁ80 °C. Protein concentrations were determined
by the Bradford method using bovine serum albumin as
standard.22
5.3. Enzyme assays
Enzyme activity was determined by monitoring the consump-
tion of PEP at 232 nm (
e
= 2.8 ꢂ 103 Mꢁ1 cmꢁ1) using a continuous
spectrophotometric assay at 30 °C.23 Assays to determine substrate
specificity contained 160
either -A5P (100 M), or
or -X5P (0.7–2 mM), or
l
D
M PEP, 0.016 U KDO8P synthase and
-L5P (1.5–4 mM), or -X5P (300 M),
D-R5P (1–3 mM; Sigma) in 50 mM BTP
D
l
L
l
D
buffer (pH 7.5) in a total volume of 1 mL. Assays were initiated
with the addition of enzyme. Also assessed was the effect of EDTA
on the activity of wild-type KDO8P synthase over time. An aliquot
of purified KDO8P synthase was removed prior to mixing the
remainder 1:1 with 2 mM EDTA. Assays (1 mL) containing
100
treated KDO8P synthase or EDTA-treated protein were initiated
with 100 -A5P. Activity of the proteins was checked after 5,
lM PEP, 50 mM BTP buffer (pH 7.5), and either 0.005 U un-
lM D
10, 15, 30, and 60 min as well as 2, 4, 8 and 24 h. For determination
of the effect of metal ion, assays were carried out in the presence of
5.2. Protein purification
MnSO4ꢃH2O (Sigma). Each 1 mL assay consisted of 100
100 -A5P, 0.006 U KDO8P synthase in 50 mM BTP (pH 7.5)
with or without 90 M MnSO4 and initiated by the addition of
A5P. Assays to determine the activity of the mutant (N59A) con-
tained 200 M PEP and 0.017 U NmeN59A in 50 mM BTP (pH 7.5)
and were initiated with 100 -A5P. All assays were preincu-
lM PEP,
lM D
The recombinant kdsA and the NmeN59A mutant genes were
both expressed using the same procedure. Overnight starter cul-
tures of E. coli BL21(DE3) cells harbouring either pT7-7-NmekdsA
or pT7-7-NmeN59A grown in 10 mL Luria Bertani (LB) broth sup-
plemented with ampicillin (0.1 mg mLꢁ1) were used the following
day to inoculate two 1 L of LB broth containing 0.1 mg mLꢁ1 ampi-
cillin. The cultures were grown at 37 °C with vigorous shaking to
l
D-
l
lM D
bated at 30 °C for 5–6 min prior to initiation and were performed
in duplicate.
an OD600 of 0.4–0.6, whereupon isopropyl thio-b-D-galactoside
5.4. Michaelis–Menten kinetics
(IPTG) was added (final concentration 1 mM). The cultures were
grown for a further 4 h at 37 °C then harvested by centrifugation
(4400g, 20 min, 4 °C). Purification of the KDO8P synthase Nme-
N59A mutant was identical to that of the NmekdsA gene product.
Cell pellets were resuspended in a minimal volume (ꢀ8 mL) of cold
lysis buffer (10 mM BTP, pH 7.5, 1 mM DTT, 200 mM KCl and
To determine the kinetics for PEP use, reaction mixtures con-
tained either, PEP (1–8)
and 300 -X5P in 50 mM BTP (pH 7.5). To determine the
parameters for the use of the aldose phosphate co-substrate assays
consisted of PEP (160 M) and either -A5P (2-300 M) or -X5P
(7.4–370 M) in 50 mM BTP (pH 7.5). Reaction mixes were prein-
lM and 80 lM D-A5P, or PEP (1–20 lM)
lM L
l
D
l
L
200
l
M PEP) and lysed by sonication on ice (15 ꢂ 15 s with
l
1 min intervals between bursts). Cellular debris were removed by
centrifugation (26,700g, 20 min, 4 °C) and the supernatant diluted
fivefold with Buffer A (10 mM BTP, pH 7.5, 1 mM DTT). The diluted
cubated for 5–6 min at 30 °C prior to initiation and brought to
the final 1 mL volume with the addition of enzyme (0.016 U N.
meningitidis KDO8P synthase). All kinetic experiments were carried
out in duplicate and the kinetic parameters were determined by
fitting the data to the Michaelis–Menten equation using Enzfitter
(Biosoft).
TM
protein was then loaded onto a Source 15Q column (Amersham;
10 ꢂ 90 mm; 4 °C) pre-equilibrated with Buffer A at a flow rate
of 2 mL minꢁ1. After washing the column with Buffer A (2 column
volumes, CV), a linear gradient from 0 to 0.11 M NaCl (5 CV) was
applied, then KDO8P synthase was eluted by a shallow linear
gradient from 0.11 to 0.18 M NaCl (6.5 CV) during which 3 mL frac-
tions were collected. Protein was analysed by SDS–polyacrylamide
gel electrophoresis (SDS–PAGE) using Laemmli’s buffer system un-
der denaturing and reducing conditions.21 Fractions that were
5.5. Preparation of D-xylose 5-phosphate
(ꢁ)-1,2-O-isopropylidene-
from -xylose 1 in two steps according to previously reported
procedures.24,25
a-D-xylofuranose 2 was prepared
D

M. Ahn et al. / Bioorg. Med. Chem. 16 (2008) 9830–9836
9835
5.6. Synthesis of (ꢁ)-1,2-O-isopropylidene–
D-diphenylphospho
(5.5 mg, 10
l
mol) were added to
D
-arabinose (0.05 g, 0.3 mmol)
xylofuranose 3
dissolved in 5 mL H2O. MgSO4 (14 mg, 0.17 mmol) and KCl
(8.4 mg, 0.13 mmol) were then added. The pH was adjusted to
(ꢁ)-1,2-O-Isopropylidene-
a
-
D
-xylofuranose 3 (0.95 g, 5 mmol)
7.6 with 10% NaOH after which 2-mercaptoethanol (10 lL,
was dissolved in 150 mL of dry CH2Cl2. Imidazole (1.36 g,
0.02 mol) and diphenylchlorophosphate (1.4 mL, 7 mmol) were
added at 0 °C after which the reaction mixture was stirred under
N2 for 6 h. The reaction was stopped by adding 10 mL H2O and
10% (v/v) HCl (10 mL). The aqueous layer was extracted three times
with CH2Cl2 (50 mL) and the combined organic extracts were
washed with saturated NaHCO3 (20 mL), H2O (2 ꢂ 20 mL), and
then saturated NaCl (20 mL). The organic layer was dried with
MgSO4 and concentrated. Purification by flash chromatography
(3:2 (v/v) ethyl acetate/hexane) gave 3 (0.65 g, 31%) as a colourless
syrup.
0.15 mmol) and pyruvate kinase (218 U) were added. The reac-
tion was initiated by the addition of hexokinase (270 U) and left
to stir at room temperature. The progress of the reaction was
followed by 31P NMR. The reaction for
D-A5P was completed
after 46 h. For the
D-lyxose 5-phosphate reaction (0.05 g,
0.3 mmol), more MgSO4 (14 mg, 0.17 mmol) and hexokinase
(109 U) were required and were added after 25 h to the reac-
tion mixture, which was then allowed to stir for a further
28 h before more MgSO4 (100 mg, 0.83 mmol), pyruvate kinase
(436 U), and hexokinase (540 U) were introduced. After an addi-
tional five days, the reaction for
in -A5P and -L5P mixtures was removed using centrifugal fil-
tration devices (10,000 MWCO; Vivaspin), and -A5P and -L5P
were purified by anion exchange chromatography as described
for -xylose 5-phosphate. Fractions containing -A5P or -L5P
D-L5P was completed. Protein
RF (hexane/EtOAc, 1:1) = 0.5.
D
D
m/z (+ve FAB) (M+H+) C20H24O8P, calcd 423.12088 found
423.1208.
D
D
1H NMR (400 MHz, CDCl3) d 7.3 (m, 10H, ArH), 5.85 (d,
J = 3.5 Hz, 1H), 4.5 (m, 2H), 4.28 (m, 2H), 4.07 (d, J = 1.9 Hz, 1H),
1.45 (s, 3H), 1.27 (s, 3H) ppm; 13C NMR (125 MHz, CDCl3) d 150.2
(d, J = 15 Hz), 130.0, 125.8 (d, J = 13.5 Hz), 120.1 (d, J = 14.3 Hz),
115.4, 105.0, 85.1, 78.8, 73.8, 65.2, 26.9, 26.2 ppm.
D
D
D
were identified by PEP consumption when incubated with N.
meningitidis KDO8P synthase or by using Bial’s reagent.27 These
fractions were pooled and freeze-dried, giving
D-A5P (73 mg,
95%) and
m/z (ꢁve ESI,
229.0119.
m/z (ꢁve ESI,
229.0115.
D-L5P (58 mg, 76%).
D
-A5P) (MꢁH)ꢁ C5H10O8P calcd 229.0113, found
5.7. Synthesis of (ꢁ)-1,2-O-isopropylidene–
D-5-phospho
xylofuranose 4
D
-L5P) (MꢁH)ꢁ C5H10O8P; calcd 229.0113, found
(ꢁ)-1,2-O-Isopropylidene-
a-D-5-diphenylphospho xylofuranose
3 (0.65 g, 1.5 mmol) was dissolved in methanol and Pt2O (0.34 g,
1.5 mmol) was added. The reaction mixture was then stirred over-
night under a complete H2 atmosphere at room temperature. Re-
moval of solvent in vacuo gave 4 (0.42 g, 100%).
5.11. KDO8P and 3-deoxy-L-gulo-octulosonate 8-phosphate
(DGO8P) preparation and isolation
Large scale enzymatic reactions were performed using N. men-
ingitidis KDO8P synthase to generate both KDO8P from PEP and
1H NMR (400 MHz, CDCl3) d 5.80 (d, 1 H, J = 3.7 Hz), 4.45 (d, 1 H,
J = 3.7 Hz), 4.19 (m, 1 H), 4.07 (d, 1 H, J = 2.8 Hz), 3.95 (m, 1 H), 3.83
(m, 1 H), 1.27 (s, 3 H), 1.11 (s, 3 H) ppm. 13C NMR (100.6 MHz,
CDCl3) d 113.0, 104.8, 84.8, 79.9, 73.9, 64.0, 25.8, 25.3 ppm.
D-A5P, and DGO8P from PEP and
L-X5P. For KDO8P,
D-A5P
(11 mg, 47.3 mol) and PEP (11 mg, 53.4
l
l
mol) were dissolved in
H2O (8 mL) and the pH was adjusted to pH 7 using 1 M NaOH.
The reaction was initiated by the addition of KDO8P synthase
(0.6 mg) and the loss of PEP at 232 nm was monitored. After 2 to
3 h, the loss of PEP had ceased, whereupon the enzyme was re-
moved as described above. The same procedure was used to gener-
5.8. Synthesis of
D
-xylose 5-phosphate 5
-5-phospho xylofuranose 4 (0.42 g,
(ꢁ)-1,2-O-Isopropylidene-
a-D
1.56 mmol) was dissolved in 15 mL of H2O and stirred for two days
ate DGO8P with L-X5P (2 mg, 9 lmol) and PEP (2 mg, 9.7 lmol)
at 50 °C. The progress of the reaction was followed by 1H NMR. The
dissolved in 6 mL H2O. The products of both reactions were puri-
fied using anion exchange chromatography (using the established
method described above but with a linear gradient of 0–1 M
NH4HCO3 (23 CV) applied to elute the phosphorylated monosac-
charides). The thiobarbituric acid assay was used to identify frac-
tions that contained the 3-deoxyaldulosonic acid product.
Fractions that tested positive were pooled and freeze-dried (giving
15 mg of KDO8P and 1.5 mg of DGO8P). The products were stored
at ꢁ80 °C prior to NMR and mass spectral analysis.
TM
D
-X5P mixture was loaded onto a Source 15Q column (Amersham;
10 ꢂ 95 mm; 4 °C) pre-equilibrated with H2O at a flow rate of
2 mL minꢁ1. After washing the column with 1.3 CV of H2O, a linear
gradient from 0 to 0.5 M NH4CO3 (20 CV) was applied which eluted
the D-X5P. Fractions containing D-X5P (determined by the loss of
PEP in an assay catalysed by E. coli DAH7P synthase) were pooled
and freeze-dried, giving 5 (0.26 g, 72%).
m/z (ꢁve ESI) (MꢁH)ꢁ C5H10O8P; calcd 229.0113 found
229.0113.
m/z (ꢁve ESI, KDO8P) Mꢁ C8H14O11P; calcd 317.0274, found
317.0277; found (MꢁH+Na)ꢁ 329.0246; found (MꢁꢁH2O)
299.0197.
5.9. Preparation of L-xylose 5-phosphate
m/z (ꢁve ESI, DGO8P) Mꢁ C8H14O11P; calcd 317.0274, found
317.0273; found (M+NaꢁH)ꢁ 329.0242; found (MꢁꢁH2O)
299.0193.
L
-X5P (0.2 g, 13% overall yield) was prepared starting with
lose (1 g, 6.67 mmol) following the same procedures for the syn-
thesis of -X5P. NMR spectra from the intermediate compounds
L-xy-
D
appeared to be same as those recorded for the synthesis of
D-X5P.
5.12. Thiobarbituric acid assay13,28
m/z (ꢁve ESI) (MꢁH)ꢁ C5H10O8P; calcd 229.0113 found
229.0110.
Aliquots (100
L H2O and 100
and heated for 1 h at 60 °C. Excess oxidising agent was removed
by the addition of 200 L sodium metaarsenite (2% w/v in 0.5 N
lL) of the reaction mixture were mixed with
50
l
l
L NaIO4 (25 mM solution in 0.125 N H2SO4)
5.10. Preparation of
phosphate26
D-arabinose 5-phosphate and D-lyxose 5-
l
HCl). Once the yellow colouration disappeared, thiobarbituric acid
(1 mL of 0.36% w/v solution adjusted to pH 9 with NaOH) was
added and the reaction mixture was heated at 100 °C for 10 min.
D
-Arabinose 5-phosphate and
D-lyxose 5-phosphate were pre-
pared using similar procedures. PEP (0.08 g, 0.34 mmol) and ATP

9836
M. Ahn et al. / Bioorg. Med. Chem. 16 (2008) 9830–9836
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J. Biol. Chem. 2004, 279, 45110.
After the sample was cooled to 25 °C the absorbance was measured
at 549 nm (
e
= 1.03 ꢂ 105 Mꢁ1 cmꢁ1).
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Acknowledgements
Meekyung Ahn is a recipient of a Massey University Vice-Chan-
cellor’s Doctoral Scholarship. We would like to thank Geoffrey
Jameson for helpful comments. This work was funded by the Mas-
sey University Research Fund and the Royal Society of New Zealand
Marsden Fund (MAU008).
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