From the
The enzyme 3-deoxy-D-manno-octulosonic acid
8-phosphate synthase catalyzes the condensation of D-arabinose
5-phosphate with phosphoenolpyruvate to give the unique 8-carbon acidic
sugar 3-deoxy-D-manno-octulosonic acid 8-phosphate
(KDO 8-P) found only in Gram-negative bacteria and required for lipid A
maturation and cellular growth. The Escherichia coli gene
kdsA that encodes KDO 8-P synthase has been amplified by
polymerase chain reaction methodologies and subcloned into the
expression vector, pT7-7. A simple one-step purification yields
200 mg of homogeneous KDO 8-P synthase per liter of cell culture.
[2-
From a chemotherapeutic point of view, the lipopolysaccharide
biosynthetic pathway in Gram-negative bacteria is an attractive target,
since mutants producing incomplete lipopolysaccharide are more
susceptible to antibiotics and less pathogenic. Since
3-deoxy-D-manno-octulosonic acid
(KDO
Biosynthesis and utilization of KDO can be envisioned as an
essential minor branched pathway in carbohydrate metabolism in
Gram-negative bacteria. A vital enzyme in this pathway is
3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO
8-P) synthase (EC 4.1.2.16). This enzyme catalyzes the condensation of
D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP)
to yield inorganic phosphate (P
Recently, both Kohen
et al.(2) and our group
(3) have reported the
steric course of the enzymatic condensation catalyzed by KDO 8-P
synthase. Like the vast majority of known PEP-utilizing enzymes, the
catalysis occurs with addition of the electrophile to the si face of PEP. This consistence in facial specificity may be
indicative of a conserved PEP binding motif. Most importantly, these
results ruled out any mechanism involving the formation of a freely
rotating sp
Knowledge of the fate of the PEP enolic oxygen atom is also critical
in evaluating the enzymatic mechanism of KDO 8-P synthase. Cleavage at
either the C-O or P-O of the PEP enolic bond can account for the
liberation of the inorganic phosphate co-produced during KDO 8-P
synthase catalysis. PEP labeled with
In this paper, we wish to report the design of an
overexpression system as well as a single-step method for the
purification of KDO 8-P synthase. The cloned enzyme has been utilized
in
The large scale (1 liter to 3 g of E. coli) expression of
KDO 8-P synthase produced 180-220 mg of homogeneous cloned enzyme
after one anion-exchange chromatographic step (center of peak). This
represented 40% of the total activity of KDO 8-P synthase present in
the crude extract. The protein purity was judged to be >98% by both
SDS-PAGE (data not shown) and size exclusion high-pressure liquid
chromatography utilizing a Synchropak GPC100 (data not shown) and had a
specific activity of 9 units/mg. Several hundred milligrams of KDO 8-P
synthase (96% pure, another 35% of the activity present in the crude
extract) may be obtained by combining the fractions from both the
leading and trailing portions of the above peak. SDS-PAGE as well as
specific activity measurements indicates that even the crude extract
could be used directly for most experiments, however, only the >98%
fraction was used in this work. The electrospray mass spectrum of KDO
8-P synthase, monomeric molecular mass of 30,842 ± 17 daltons,
and the experimentally determined NH
In PEP-utilizing enzymes, oxygen dynamics are very important
in distinguishing between hypothesized mechanisms. Some of these
enzymes, such as KDO 8-P synthase, yield inorganic phosphate
(P
It has been established that
When [2-
Since C-O bond cleavage of the PEP enolic bond
precludes the bridging oxygen from assuming the role of the anomeric
oxygen of KDO 8-P, the origin of the anomeric oxygen needed to be
determined. To this end, [2-
KDO
8-P synthase catalysis has been shown to occur stereospecifically by
the si face addition of C-3 of PEP upon the re face
of the carbonyl carbon of A 5-P with no incorporation of solvent
protons which was interpreted to mean that no freely rotating methyl
group is formed in the reaction pathway
(2, 3) . The
results of the
We thank Dr. Jack E. Dixon of the Biological Chemistry
Department for allowing J. C. C. to participate in this work. We thank
the Department of Chemistry at the University of Michigan for access to
their AMX500 spectrometer.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C,
O]Phosphoenolpyruvate (PEP)
was prepared by first, exchange of
[2-
C]-3-bromopyruvate with
H
O followed by reaction of the
labeled bromopyruvate with trimethylphosphite. The fate of the enolic
oxygen in this multilabeled PEP, during the course of the KDO 8-P
synthase-catalyzed reaction with D-arabinose 5-phosphate, was
monitored by
C and
P NMR spectroscopy. The
inorganic phosphate formed during the reaction was further analyzed via
mass spectral analysis of its trimethyl ester derivative. The
C NMR spectrum of an incubation mixture of
[2-
C]PEP and D-arabinose 5-phosphate in
H
O in the presence of KDO 8-P
synthase was also recorded. [2-
C]KDO 8-P was
utilized to determine the extent of nonenzymatic incorporation of
O into the C-2 position of KDO 8-P. The results indicate
that the enolic oxygen of the PEP is recovered with the inorganic
phosphate, and the C-2 oxygen of KDO 8-P originates from the solvent,
H
O.
(
)
;
2-keto-3-deoxy-D-manno-octulosonic acid) is a
site-specific molecule found only in Gram-negative organisms and is
required for lipid A maturation and cellular growth, the inhibition of
its production would be an excellent chemotherapeutic goal
(1) .
Inhibitors of the enzyme(s) responsible for the biosynthesis of KDO,
then, would represent a novel class of antibiotics since no existing
drugs work by disrupting synthesis of lipopolysaccharide.
) and KDO 8-P. KDO 8-P is
dephosphorylated by the next enzyme in the pathway to give KDO which is
subsequently activated to cytidine
5`-monophosphate-3-deoxy-D-manno-octulosonate and
transferred to a lipopolysaccharide precursor
(1) . KDO 8-P
synthase is a member of a family of enzymes including
3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P)
synthase (EC 4.1.2.15) and N-acylneuraminate-9-phosphate
synthase (EC 4.1.3.20) which catalyzes the condensation of PEP with a
phosphorylated sugar to produce a new phosphorylated 3-deoxy-
-keto
sugar acid three carbons longer. Understanding the mechanism by which
these enzymes catalyze this aldol condensation would be of great
importance in the design of selective inhibitors for each of these
biologically important 3-deoxycarbohydrates.
center at C-3 of PEP during catalysis.
O in the enolic
oxygen has classically been used to distinguish between these two types
of bond cleavage. Studies previously published by Hedstrom and Abeles
(4) on the fate of the PEP enolic oxygen during KDO 8-P synthase
catalysis, using such an
O-labeled PEP, were accomplished
utilizing only partially purified enzyme extracts containing just 10%
KDO 8-P synthase. In addition, certain quantitative aspects of the
investigation, such as the amount of A 5-P used as substrate and the
amount of KDO 8-P formed relative to the P
generated, were
not reported.
C and
P NMR studies to establish the fate
of the enolic oxygen of PEP during KDO 8-P synthase catalysis and to
identify the origin of the anomeric oxygen of enzymatically derived KDO
8-P.
Materials
The chemicals used were of reagent grade or of the highest
purity commercially available and were not further purified.
Q-Sepharose, ammonium sulfate, D-arabinose 5-phosphate,
phosphoenolpyruvate (unlabeled), 3-deoxyoctulosonic acid,
dithiothreitol, and Tris were purchased from Sigma. Sodium
[2-C]pyruvate (99%
C) and
H
O (un-normalized; 98%
O, 90%
H) were purchased from Cambridge
Isotope Laboratory. Restriction and DNA modifying enzymes were from
Boehringer Mannheim. The Escherichia coli strains BL 21 and BL
(DE 3) were obtained from Novagen. The thermal cycling was performed
using an MJR Research Thermal cycler. Oligonucleotides were synthesized
by the University of Michigan, Biomedical Research Resources Core
Facility, and at Warner-Lambert Parke-Davis. DNA sequencing,
NH
-terminal protein sequencing, and total amino acid
content as well as electrospray mass spectral analysis were performed
by the University of Michigan, Biomedical Research Resources Core
Facility. The Perkin-Elmer GeneAmp kit was used for the PCR reaction
except recombinant pfu DNA polymerase (Stratagene Cloning
Systems, Buffer 1) was substituted for the Taq DNA polymerase.
Promega DNA and PCR purification kits were utilized. Mass spectral
analyses of small molecular weight compounds were performed at The
Michigan State University Biochemistry Instrument Facility. The Kohara
phage was provided by Dr. Rowena G. Matthews of the Department of
Biological Chemistry at the University of Michigan.
PCR Cloning of kdsA
Two primers were constructed to correspond to the 5` and 3`
ends of the open reading frame identified as the kdsA gene.
The 5` sequence was GATTCTAGAATTCATATGAAACAAAAAGTGGTT. The 3`
sequence was AAAGATCTTACTTGCTGGTATCCA. The forward primer
incorporated an NdeI (underlined) and the reverse primer a
BglII (underlined) site for cloning into the expression
vector, pT7-7
(5) . The kdsA gene was amplified
(30 cycles) from Kohara phage 4D10 by PCR
(6) . The
annealing temperature was 45 °C. The PCR-amplified DNA was
sequentially digested with NdeI and BglII and
directionally cloned into NdeI/BamHI-restricted
pT7-7. Recombinants from TG1 cells were obtained and screened by
restriction analysis. Appropriate plasmid DNA
(pT7-7/kdsA) was used to transform competent BL 21 (DE
3) cells. The correct sequence
(7) of the cloned kdsA gene was confirmed by automated sequencing on an Applied
Biosystems 373A automated DNA sequencer utilizing dye-labeled dideoxy
nucleotides, Taq polymerase and double-stranded DNA, purified
by the Promega PCR purification kit. Both strands of the plasmid DNA
were sequenced.
Preparation and Purification of Recombinant KDO 8-P
Synthase
Cell Culture
E. coli BL 21 (DE 3)
harboring the plasmid pT7-7/kdsA, was grown to an
A = 0.6 in 5 ml of 2xTY media. The
preparative media (2xTY, 50 µg/ml ampicillin) was inoculated with
1000 µl of the above culture per 200 ml of media. The cultures were
incubated at 37 °C with vigorous shaking (300 rpm) in 1-liter
baffled flasks (200 ml/flask) to an A
=
0.6-0.8 and 400 µl of 0.2 mM
isopropyl-1-thio-
[D-galactopyranoside was added. The
incubation (37 °C, 300 rpm) was continued for 4 h.
Preparation of Crude Extract
Cells from the above
culture were harvested by centrifugation (4,500 g, 15
min) and the total wet weight of the cells was measured (3.4 g/liter of
2xTY). The cell pellet was suspended in 20 mM Tris-HCl (pH
7.4) buffer (1 g/5 m). This step and all subsequent purification steps
were performed at 4 °C and all buffers contained 0.2 mM
dithiothreitol. The cells were disrupted by 2 min of sonication, with
cooling (4
30 s pulses with a 30 s delay between pulses) and
centrifuged at 25,000
g for 20 min. The supernatant
was removed and the pellet resuspended in Tris-HCl buffer as described
above and the whole process repeated. The supernatants were combined
for the next step.
Anion Exchange Chromatography
The pH of the
cell-free extract was adjusted to 7.4 with 1 M Tris base and
filtered through a 0.22-µm sterile filter. The cell-free extract
was divided into three portions. Each portion of the cell-free extract
was applied to an anion exchange column (1.5 70-cm column
packed with Q-Sepharose) equilibrated with 20 mM Tris-HCl (pH
7.4) buffer containing 75 mM KCl. The column was first washed
with 200 ml of equilibration buffer and then eluted with 1 liter of a
linear gradient of 100-400 mM KCl in 20 mM
Tris-HCl (pH 7.4) buffer. The flow rate was kept at 1 ml/min. Fractions
(13.5 ml) containing KDO 8-P synthase, determined by the periodate-TBA
assay (8) and SDS-PAGE, were pooled and subjected to concentration via
lyophilization. The concentrated enzyme solutions from three individual
runs were combined and dialyzed overnight against 5 mM
potassium phosphate (pH 7.2) buffer (3
1000 ml).
Assay Procedures
KDO 8-P Synthase Assay
Assay mixtures contained
0.1 M Tris acetate (pH 7.5), 3 mM PEP, 3 mM
A 5-P, and enzyme, in a final volume of 150 µl. After incubation
for 10 min at 37 °C, the reaction was terminated by the addition of
150 µl of 10% trichloroacetic acid and centrifuged to remove the
protein. An aliquot of the incubation mixture was used to determine
either the amount of KDO 8-P produced using the periodate-TBA assay
reported for KDO 8-P synthase by Ray
(8) , or the amount of
P produced using the method of Lanzetta et
al.(9) . Both methods give similar results. One unit of
activity is defined as 1 µmol of KDO 8-P of P
released
per min at 37 °C.
Protein Assay
The protein concentrations of enzyme
fractions were determined using the Bio-Rad protein assay (Bio-Rad)
with bovine serum albumin as standard. Based on numerous total amino
acid composition analyses, the Bio-Rad protein assay overestimates KDO
8-P synthase by a factor of 1.3.
Polyacrylamide Gel Electrophoresis
Electrophoretic analyses were performed utilizing 12%
denaturing gels in the discontinuous Laemmli buffer system with a
Bio-Rad Mini-Protean II. Gels were stained using 0.25% Coomassie
Brilliant Blue R-250.
Amino Acid Composition and Amino-terminal Sequence
Analysis
The total amino acid content of the recombinant KDO 8-P
synthase was determined by the University of Michigan, Biomedical
Research Resources Core Facility utilizing an Applied Biosystems 420
analyzer equipped with a model 130 HPLC unit. The first 10
amino-terminal amino acids were sequenced by the University of
Michigan, Biomedical Research Resources Core Facility via an Applied
Biosystems 473 analyzer.
Molecular Weight Determination
The molecular weight of recombinant KDO 8-P synthase was
determined by electrospray mass spectroscopy performed by the
University of Michigan, Biomedical Research Resources Core Facility,
utilizing a Vestec electrospray mass spectrometer.
Mass Spectrometry of Small M
A gas chromatograph-mass spectrometer in the electron
ionization mode was used at the Michigan State University Biochemistry
Instrument Facility for the analysis of trimethyl phosphate.
Compounds
Synthesis of [2-13C]Phosphoenolpyruvate
To a suspension of sodium
[2-C]pyruvate (1.0 g, 9 mmol) in CCl
(25 ml), was added 1.1 eq of concentrated HCl and 1.0 eq of
bromine
(10) . The reaction was stirred at 25 °C until the
reaction mixture was colorless. The reaction was then filtered to
remove NaCl and the solvent removed by rotary evaporation. The
[2-
C]3-bromopyruvic acid was taken up into
anhydrous diethyl ether and converted into dimethyl
[2-
C]phosphoenolpyruvate by treatment with
trimethyl phosphite under Perkow reaction conditions
(11) . The
title compound was achieved as the monocyclohexylamine salt (m.p.
143-144 °C; lit. (11) 143-146 °C) by the method
reported by Clark and Kirby
(12) .
Synthesis of
[2-
The [2- C,
O]Phosphoenolpyruvate
C]3-bromopyruvic acid
synthesized as above was converted into
[2-
C,
O]3-bromopyruvic acid by the
exchange method of Bondinell et al.(13) utilizing a
mixture of
H
O (un-normalized; 98%
O, 90%
H) (350 µl) and H
O (120
µl). The [2-
C,
O]3-bromopyruvic
acid was then converted into the title compound as described above
(m.p. 143-144 °C; lit. 143-146 °C)
(12) . The
PEP obtained from the
3-bromo-[2-
C,
O]pyruvic acid
reaction contained 70%
O label in the enolic oxygen. This
was determined by integration of the C-2 resonance due to
[2-
C,
O]PEP and the
O-shifted carbon resonance due to
[2-
C,
O]PEP, in the subspectra
created by multiple-quantum filtration of the
C NMR
spectra
(14) .
Enzymatic Conversion of
[2-
KDO 8-P synthase (1 unit) was added to a 10-ml Erlenmeyer
flask containing the following:
[2- C]Phosphoenolpyruvate to
[2-
C]3-Deoxyoctulosonate 8-Phosphate
C]phosphoenolpyruvate monocyclohexylamine
salt (6.7 mg, 0.025 mmol), A 5-P disodium salt (7.0 mg, 0.025 mmol),
Tris-HCl (pH 7.5) (0.5 mmol), and H
O, in a final volume of
2.5 ml. This reaction mixture was incubated at 37 °C for 2 h in a
shaking water bath. The reaction mixture, unquenched, was loaded
directly onto a 1.5
27-cm anion exchange column (Bio-Rad
AG-MP1, Cl
form), washed with 100 ml of
H
O, and eluted with 500 ml of a linear gradient of
0-400 mM NaCl. Two periodate-TBA positive peaks were
obtained, the first at 85 mM, KDO (5%), and the second at 160
mM, KDO 8-P (95%). The fractions containing the KDO 8-P were
pooled, freeze-dried, reconstituted in 2 ml of H
O, and
desalted on a 2
60-cm Bio-Gel P-2 column. The periodate-TBA
positive/AgCl negative fractions were pooled and freeze-dried to give
the desired [2-
C]KDO 8-P.
Nuclear Magnetic Resonance Methods
Proton-decoupled
P NMR
P NMR spectra were recorded on a Bruker AMX500
spectrometer (11.75 T) at a probe temperature of 298 K, tuned to 202.4
MHz, using 5-mm high resolution NMR tubes. Spectra were obtained with a
spectral width of 2000 Hz, 1.0 s relaxation delay, and 32,768 complex
points in the time domain using simultaneous detection of real and
imaginary components. The GARP sequence was used for heteronuclear
decoupling. The time domain data were apodized with an exponential (0.5
Hz for X-nuclei) prior to zero-filling followed by Fourier
transformation. Chemical shifts were reported relative to an external
sample of 10 mM inorganic phosphate (0.0 ppm) in 200
mM Tris-HCl (pH 7.5) containing 10 mM EGTA and 10%
H
O.
Proton-decoupled
C NMR
C NMR spectra were recorded on a Bruker AMX500
spectrometer (11.75 T) at a probe temperature of 298 K, tuned to 125.7
MHz, using 5-mm high resolution NMR tubes. Spectra were obtained with a
spectral width of 25,000 Hz, 1.0 s relaxation delay, and 32,768 complex
points in the time domain using simultaneous detection of real and
imaginary components. The GARP sequence was used for heteronuclear
decoupling. The time domain data were apodized with an exponential (0.5
Hz for X-nuclei) prior to zero-filling followed by Fourier
transformation. Chemical shifts are reported in parts per million
relative to tetramethylsilane. Dioxane in sample buffer is used as an
external reference (67.3 ppm).
KDO 8-P Synthase Reaction in
[
The reaction mixtures contained the following in 0.5 ml of
200 mM Tris-HCl (pH 7.5): 10 mM A 5-P, 10 mM
[2- O]H
O
C,
O]PEP (99%
C, 70%
O), 10 mM EGTA, 10%
H
O
for field frequency lock, and 0.2 units of KDO 8-P synthase. All
components of the reaction mixture except KDO 8-P synthase were
equilibrated at 25 °C. After taking a control spectrum the reaction
was initiated by the addition of KDO 8-P synthase. Data accumulation
began 2-4 min after addition of the enzyme.
KDO 8-P Synthase Reaction in
[2- H
O
C]PEP (5 µmol), A 5-P (5
µmol), and EGTA (5 µmol) were dissolved in 200 mM
Tris-HCl (500 µl) (pH 7.5) and lyophilized to dryness.
H
O (350 µl) and H
O
(120 µl) were added to the dry powder, and the solution transferred
to a 5-mm NMR tube. All components of the reaction mixture except KDO
8-P synthase were equilibrated at 25 °C. After taking a control
C NMR spectrum the reaction was initiated by the addition
of KDO 8-P synthase (0.2 units; 30 µl). Data accumulation began
2-4 min after addition of the enzyme.
Purification of P
from Enzymatic
Reaction
Gel Filtration Chromatography
The reaction
mixtures which utilized [2-C,
O]PEP
as a substrate were combined at the conclusion of the NMR experiments
and loaded onto a 2
66-cm Bio-Gel P-2 column equilibrated with
H
O at 25 °C. The column was eluted with H
O
and the fractions (8 ml) assayed for P
, A 5-P, and KDO 8-P.
The fractions containing P
were well resolved from KDO 8-P
but did contain some A 5-P. Fractions containing P
without
A 5-P were pooled and lyophilized. Additional P
fractions
containing A 5-P were combined and lyophilized separately. The total
amount of P
obtained was equivalent to the amount of KDO
8-P formed.
Precipitation of P
The lyophilized
P as
MgNH
PO
fractions containing A 5-P were dissolved in 3 ml of
H
O and 0.5 ml of magnesia mixture (2.7 mmol of
MgCl
, 18.7 mmol of NH
Cl, 15 mmol of
NH
OH in 10 ml of H
O) added. The pH of the
mixture was then adjusted to pH 9 with 2.5 M NH
OH
and allowed to stand at 4 °C for 12 h. The MgNH
PO
precipitate was collected by centrifugation, washed with 3 ml of
cold 3.7 M NH
OH. The base-washed precipitate was
collected by centrifugation, and dried in vacuo.
Cation Exchange Chromatography
The
MgNHPO
precipitate was then combined with the
lyophilized pure P
fractions (from the gel filtration
column) and dissolved in 1 ml of H
O containing a small
amount of Bio-Rad AG 50W-X8 resin (H
form) to aid in
solubilization. The resin suspension was transferred to a 1
10-cm Bio-Rad AG 50W-X8 resin (H
form) column and
eluted with H
O. The P
positive fractions were
combined, the pH adjusted to 4.1 with 0.1 N KOH, and
lyophilized to yield potassium phosphate (
6 µmol).
Conversion of Biosynthetic P
To the lyophilization vessel containing P to Its
Trimethyl Ester
was added 100 µl of 95% ethanol and a drop of concentrated
HCl. Methylation was accomplished by the dropwise addition of a fresh
solution of diazomethane in ether to the lyophilization vessel. The
solvent was then removed by a stream of dry nitrogen.
Overproduction and Purification of E. coli kdsA
The T7 polymerase-dependent expression system pT7-7 was
used to overexpress KDO 8-P synthase in E. coli BL 21 (DE 3).
-terminal amino acid
sequence, MKQKVVSIGD, are both consistent with the molecular mass and
amino acid sequence predicted from the DNA sequence of kdsA,
respectively. The total amino acid content was also consistent with
theoretical values.
Oxygen Transfer during KDO 8-P Synthase Catalysis
The fate of the PEP enolic oxygen during enzymatic catalysis
and the origin of the anomeric oxygen of KDO 8-P were investigated by
one-dimensional isotopic-shifted C and
P NMR.
Fate of the PEP Enolic Oxygen
Fig. 1
depicts the
proton-decoupled P NMR spectra of a KDO 8-P synthase
reaction mixture using doubly labeled
[2-
C,
O]PEP (70%
O and
99%
C) as a substrate. Spectrum ashows
the reaction mixture before the addition of enzyme. The phosphorus
resonance of the
and
anomers of A 5-P (60:40) can be seen
as two singlets centered at 1.3 ppm. The PEP phosphorus resonance
centered at -3.1 ppm is a multiplet consisting of two pairs of
C coupled doublets arising from either
O or
O in the enolic position, and further isotopic shifting of
each of these signals due to vinylic deuterium incorporation during the
O labeling process. Seventy percent of the PEP is
O labeled in the enolic position as determined by
integration of the multiple-quantum filtered
C NMR
spectra
(14) . Some nonspecific substrate degradation from both
PEP and A 5-P resulting in P
contamination can be seen at 0
ppm consisting of two singlets of approximately equal intensities of
which the downfield singlet represents
[
O
]P
and the upfield
singlet represents
[
O
,
O]P
.
Spectra b, c, and d depict the enzymatic reaction at
various time intervals after the addition of KDO 8-P synthase. The
insert above spectrum dis an expansion of the P
region from the difference spectrum generated by substrating
spectrum dfrom a (d-a). The
-pyranose anomer of KDO 8-P can be seen growing in at 1.91 ppm
with a subsequent increase in P
and a decrease in substrate
intensities. The
-pyranose anomer of KDO 8-P was chosen for
monitoring because it is the most abundant and appears first. The
-pyranose as well as the
- and
-furanose anomers give
identical results (two of the three resonances can be seen on either
side of the major
-pyranose resonance). A closer examination of
the
P resonances due to P
reveal that it is
composed of a majority of
O labeled P
as would
be expected in the case of C-O bond cleavage of the PEP enolic bond.
The ratio of
[
O
]P
/[
O
,
O]P
in the difference spectra, spectra d-a(shown as
an expanded insert above spectrum din Fig. 1),
is 32/68 as determined by the integration of the respective phosphorus
resonances.
Figure 1:
P NMR spectra of the KDO
8-P synthase incubation mixture using
[2-
C,
O]PEP as a substrate.
a, proton-decoupled
P NMR spectrum of A 5-P (10
mM) and [2-
C,
O]PEP (99%
C, 70%
O; 10 mM) in 200 mM
Tris-HCl in 10%
H
O containing EGTA (10
mM) at pH 7.5. b-d, proton-decoupled
P
NMR spectra obtained after the addition of KDO 8-P synthase (0.2 units)
to the incubation mixture in a. Spectrum b was
obtained 8 min after the addition of KDO 8-P synthase (37 scans);
spectrum c was obtained after 28 min (166 scans); and
spectrum d was obtained after 58 min (358 scans). The new
resonances appearing downfield of the A 5-P phosphorus resonances
correspond to the phosphorus resonances of the various KDO 8-P
configurational isomers.
From a reaction identical to that in Fig. 1,
Fig. 2
shows the regions of the proton-decoupled C
NMR spectra corresponding to the C-2 resonance of
[2-
C,
O]PEP (149.95 ppm) and again
only that of the C-2 resonance of the
-pyranose anomer of
KDO 8-P (96.85 ppm) although the other isomers are present.
Spectrum ashows the reaction mixture before the
addition of enzyme. The complexity of the
C C-2 PEP
resonance is due to the
P scalar coupling and the isotopic
shielding effects from the presence of
H and
O
(14) . Spectra b-ddepict the
enzymatic reaction at various time intervals after the addition of KDO
8-P synthase. The minor peak seen slightly upfield of the major
resonance at 96.85 ppm is due to the C-3 deuterium isotopic shielding
effect upon the anomeric carbon of KDO 8-P and corresponds to the
amount of deuterium seen in the PEP substrate.
Figure 2:
C NMR spectra of the KDO 8-P
synthase incubation mixture using
[2-
C,
O]PEP as a substrate.
a, proton-decoupled
C NMR spectrum of A 5-P (10
mM) and [2-
C,
O]PEP (99%
C, 70%
O; 10 mM) in 200 mM
Tris-HCl in 10%
H
O containing EGTA (10
mM) at pH 7.5. b-d, proton-decoupled
C
NMR spectra obtained after the addition of KDO 8-P synthase (0.2 units)
to the incubation mixture in a. Spectrum b was
obtained 15 min after the addition of KDO 8-P synthase (226 scans);
spectrum c was obtained after 25 min (463 scans); and
spectrum d was obtained after 41 min (810 scans). The new
resonance at 96.85 ppm corresponds to C-2 of the newly formed KDO 8-P
(
-pyranose). The minor peak resonating slightly upfield of this
resonance is due to the C-3 deuterium isotopic shielding effect upon
the anomeric carbon of KDO 8-P.
Quantitative mass
spectral analysis of the trimethyl phosphate derivative of the
enzymatically obtained P from the above reactions contained
67.4%
O label and gave a fragmentation pattern
corresponding to that which was described by Banerjee et
al.(15) .
Origin of the Anomeric Oxygen of KDO 8-P
A
C NMR experiment with [2-
C]PEP as
substrate in
H
O (70%
O, 64% D) was performed to determine the origin of the
anomeric oxygen of KDO 8-P and to confirm the C-O enolic bond cleavage
of PEP during enzymatic catalysis. Fig. 3shows a series of
C NMR spectra of a KDO 8-P synthase reaction mixture
containing [2-
C]PEP in 70%
H
O and 30%
H
O. Only those regions corresponding to the
C-2 resonance of [2-
C]PEP (149.95 ppm) and that
of the C-2 resonance of only the
-pyranose anomer of KDO
8-P (96.85 ppm) are depicted. Spectrum ashows the
reaction mixture before the addition of enzyme. Spectra b-ddepict the enzymatic reaction at various time intervals after the
addition of KDO 8-P synthase. Analysis of the
C resonance
corresponding to the anomeric carbon (96.85 ppm) reveals the presence
of two peaks indicative of incorporation of
O from solvent
into the anomeric position of KDO 8-P. Integration of the peaks reveals
the ratio of the downfield singlet to the upfield singlet in
spectra b-d to be 32/68 which represents the ratio of
[2-
C,
O]KDO
8-P/[2-
C,
O]KDO 8-P. In a control
experiment, [2-
C]KDO 8-P was incubated under
identical solvent, concentration, pH, time, and temperature conditions
as described above without the addition of enzyme or substrates. No
nonenzymatic exchange of
O into the anomeric position
could be detected by
C NMR over the duration of the
experiment.
Figure 3:
C NMR spectra of the KDO 8-P
synthase incubation mixture using [2-
C]PEP as a
substrate. a, proton-decoupled
C NMR spectrum of
A 5-P (10 mM) and [2-
C]PEP (99%
C; 10 mM) in 200 mM Tris-HCl in 70%
[
H
O] containing EGTA (10
mM) at pH 7.5. The doublet at 149.95 ppm corresponds to the
C-2 resonance of PEP. b-d, proton-decoupled
C NMR
spectra obtained after the addition of KDO 8-P synthase (0.2 units) to
the incubation mixture in a. Spectrum b was obtained
15 min after the addition of KDO 8-P synthase (226 scans); spectrum
c was obtained after 28 min (561 scans); and spectrum d was obtained after 72 min (1586 scans). The new resonances seen at
96.85 ppm are assigned to C-2 of the newly formed KDO 8-P
(
-pyranose), of which the major resonance corresponds to the
anomeric carbon species bearing
O while the minor
resonance corresponds to the anomeric carbon species bearing
O. The absorbance scale is the same for both sections of
the spectra.
) as one of their products. Production of P
can be accounted for by either C-O or P-O cleavage of the PEP
enolic bond. Distinguishing between these two types of bond cleavage is
mechanistically important and can only be ascertained with the use of
oxygen isotopes. Nucleophilic attack can take place either at
phosphorus or one of the vinyl carbons. Nucleophilic attack at
phosphorus, as seen in PEP carboxylase
(16) results in P-O bond
cleavage and is coupled to the addition of an electropositive atom to
C-3 of PEP. Among those enzymes which have been shown to catalyze C-O
bond cleavage of the PEP enolic bond are DAH 7-P synthase,
5-enolpyruvylshikimate-3-phosphate synthase, and UDP-GlcNAc
enolpyruvate transferase
(17, 18, 19) . In these
enzymes, nucleophilic attack upon C-2 of PEP is coupled with the
addition of an electrophile at C-3. P
is produced by either
elimination, in the case of 5-enolpyruvylshikimate-3-phosphate synthase
and UDP-N-acetylglucosamine enolpyruvate transferase, or
nucleophilic displacement, as in the case of DAH 7-P synthase with the
resulting cleavage of the C-O enolic bond of PEP. The classical
methodology used to determine the fate of the PEP enolic oxygen is to
utilize PEP labeled with
O in the enolic oxygen as the
substrate in the enzymatic reaction
(13) . The biosynthetic
products are then isolated from the enzymatic mixture, derivatized, and
subjected to mass spectral analysis to determine the
O
content.
O has a
significant one-bond nuclear shielding effect upon carbon and
phosphorus atoms to which it is directly bonded, resulting in an
observable upfield shift in the NMR resonance of such
nuclei
(20) . Thus, isotopic-shifted heteronuclear NMR has been
used to study oxygen transfer during enzymatic catalysis (21-23).
This methodology has been adapted in the present paper to investigate
the fate of the PEP enolic oxygen during KDO 8-P synthase catalysis,
previously studied by Hedstrom and Abeles
(4) using mass
spectral analysis, and to determine the origin of the anomeric oxygen
of KDO 8-P. The previous mass spectral study utilized an enzyme mixture
that only contained 10% KDO 8-P synthase having a specific activity of
1.15 µmol/min/mg versus a specific activity of 9
µmol/min/mg for the enzyme used in the present study. In addition,
the amount of KDO 8-P or its decomposition product, KDO, formed in the
reaction from which the
O-labeled phosphate was isolated,
was not reported. It is possible that the phosphate analyzed for
O content could have been contaminated by phosphate from
other modes of PEP degradation. This NMR method of analysis has the
advantage of being able to monitor the formation of products during the
course of the reaction and eliminates the necessity of isolating the
products from the enzymatic mixture thus avoiding the possible loss of
O label during isolation and derivatization processes.
C,
O]PEP and A 5-P are
incubated in the presence of KDO 8-P synthase, the
O label
is liberated in the form of P
. The phosphorus resonance of
the
O-labeled P
([
O,
O
]P
)
is observed in the
P NMR spectra of the reaction mixture
as a major isotopically shifted singlet positioned 0.025 ppm upfield of
the minor nonlabeled P
([
O
]P
) phosphorus
resonance at 0.0 ppm (Fig. 1). The amount of
[
O,
O
]P
, as
determined by integration of the
[
O
]P
and
[
O,
O
]P
resonances in the
P NMR spectrum, corresponds to 68%
of the total P
produced from the reaction. This percentage
of [
O,
O
]P
accounts for, within the error of the NMR integration (±
5%), the percentage of
O present in the labeled PEP
substrate. The percentage of
[
O,
O
]P
was
corroborated by mass spectral analysis of the trimethyl phosphate
derivative of P
isolated from the reaction mixture. The
C NMR spectra of an identical reaction mixture
(Fig. 2) are devoid of an
O isotopically shifted KDO
8-P anomeric carbon resonance denoting the absence of a directly
attached
O and confirming C-O bond cleavage of the PEP
enolic bond.
C]PEP and A 5-P were
incubated with KDO 8-P synthase in water containing 30%
H
O and 70%
H
O to ascertain whether or not
water was the source of the anomeric oxygen. The KDO 8-P produced from
this incubation contained 68%
O in the anomeric oxygen as
determined by integration of the [
O]C-2 and
[
O]C-2 resonances of the
-pyranose anomer
in the
C NMR spectra of the reaction mixture
(Fig. 3). It has been shown that the anomeric oxygen atom of KDO
exchanges with water (t = 35 h at pH 7.2)
(23) .
It was necessary, therefore, to determine the extent of nonenzymatic
exchange of solvent oxygen into the anomeric position of KDO 8-P over
the time course of the above enzymatic reaction. In the control
reaction [2-
C]KDO 8-P was incubated in 30%
H
O, 70%
H
O under identical buffer, time
and temperature conditions as in the enzymatic reaction. No exchange of
O into the anomeric position could be seen by
C NMR over the duration of the enzymatic reaction.
O experiments presented here indicate that,
following nucleophilic attack at C-2 of PEP with condensation of C-3 of
PEP upon the carbonyl carbon of A 5-P, P
is liberated by
C-O bond cleavage at C-2 with the incorporation of solvent oxygen in
the anomeric position of product. The initiating nucleophilic attack at
C-2 of PEP may come from water, or from either the C-2 or C-3 hydroxy
groups of A 5-P. Attack of water at C-2 of PEP would yield the
open-chain C-2 phosphorylated tetrahedral species seen in
Fig. S1
. Displacement of the C-2 phosphate group could then occur
either by (a) elimination with the formation of the C-2 keto
form of KDO 8-P, (b) S
2 displacement by
the C-5 hydroxy group resulting in KDO 8-P in a furanose configuration,
or (c) S
2 displacement by the C-6 hydroxy
group resulting in KDO 8-P in a pyranose configuration (Fig. S1).
Nucleophilic attack by a secondary hydroxyl group upon C-2 of PEP has
been shown to occur in 5-enolpyruvylshikimate-3-phosphate synthase
(24) and in UDP-N-acetylglucosamine enolpyruvate
transferase
(25) . Fig. S2shows the species that would
result from an initial nucleophilic attack by the C-2 or C-3 hydroxy
groups of A 5-P upon C-2 of PEP and the subsequent condensation of C-3
of PEP upon the carbonyl carbon of A 5-P. In this case the C-2
phosphorylated tetrahedral intermediate exists as either: (a)
a furanose (attack by C-2 hydroxyl) or (b) a pyranose (attack
by C-3 hydroxyl). Displacement of phosphate to form KDO 8-P could then
occur from (c) S
2 attack by water or
(d) oxonium formation of the ring oxygen followed by the
addition of water to the anomeric carbon. Alternate PEP and A 5-P
analogues are currently being synthesized and utilized to distinquish
between these various mechanistic pathways.
Figure S1:
Scheme 1. Mechanism 1 in which a water
molecule attacks at C-2 of PEP.
Figure S2:
Scheme 2. Mechanism 2 in which either the
C-2 or C-3 hydroxyl group of A 5-P attacks at the C-2 of
PEP.
, inorganic phosphate; UDP-GlcNAc,
uridine-5`-diphospho-N-acetyl-2-amino-2-deoxyglucose; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.