©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR (*)

Garry D. Dotson (1)(§), Rajesh K. Dua (1), James C. Clemens (2), E. Wrenn Wooten (1) (3), Ronald W. Woodard (1)(¶)

From the (1) Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, Department of (2) Biochemistry and (3) Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109-1065

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-C,O]Phosphoenolpyruvate (PEP) was prepared by first, exchange of [2-C]-3-bromopyruvate with HO 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 HO 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, HO.


INTRODUCTION

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() ; 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.

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) 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.

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 center at C-3 of PEP during catalysis.

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 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.

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 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.


EXPERIMENTAL PROCEDURES

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 HO (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 MCompounds

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.

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- C, O]Phosphoenolpyruvate

The [2-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 HO (un-normalized; 98% O, 90% H) (350 µl) and HO (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- C]Phosphoenolpyruvate to [2- C]3-Deoxyoctulosonate 8-Phosphate

KDO 8-P synthase (1 unit) was added to a 10-ml Erlenmeyer flask containing the following: [2-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 HO, 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 HO, 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 HO, 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

P NMR

Proton-decoupled 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% HO.

C NMR

Proton-decoupled 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 [ O]H O

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-C,O]PEP (99% C, 70% O), 10 mM EGTA, 10% HO 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 H O

[2-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. HO (350 µl) and HO (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 HO at 25 °C. The column was eluted with HO 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 as MgNH PO

The lyophilized P fractions containing A 5-P were dissolved in 3 ml of HO and 0.5 ml of magnesia mixture (2.7 mmol of MgCl, 18.7 mmol of NHCl, 15 mmol of NHOH in 10 ml of HO) added. The pH of the mixture was then adjusted to pH 9 with 2.5 M NHOH and allowed to stand at 4 °C for 12 h. The MgNHPO precipitate was collected by centrifugation, washed with 3 ml of cold 3.7 M NHOH. 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 HO 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 HO. 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 Its Trimethyl Ester

To the lyophilization vessel containing P 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.


RESULTS

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).

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-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% HO 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% HO 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 HO (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% HO and 30% HO. 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% [HO] 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.




DISCUSSION

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) 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.

It has been established that 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.

When [2-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.

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-C]PEP and A 5-P were incubated with KDO 8-P synthase in water containing 30% HO and 70% HO 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% HO, 70% HO 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.

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 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) S2 displacement by the C-5 hydroxy group resulting in KDO 8-P in a furanose configuration, or (c) S2 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) S2 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.




FOOTNOTES

*
This work was partially supported by U. S. Public Health Service Grant GM 42544 (to R. W. W.), Biomedical Research Support Grant 2S07RR0557-28 and the Upjohn Research Endowment Fund administered by the College of Pharmacy (to R. W. W.). Acknowledgment is made to the Donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research (to E. W. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by the National Institutes of Health Training Grant T32 GM07767 and a Merit Fellowship administered by the University of Michigan Rackham Graduate School.

To whom correspondence should be addressed.

The abbreviations used are: KDO 8-P, 3-deoxyoctulosonate 8-phosphate; A 5-P, D-arabinose 5-phosphate; DAH 7-P, 3-deoxyheptulosonate 7-phosphate; PEP, phosphoenolpyruvate; P, inorganic phosphate; UDP-GlcNAc, uridine-5`-diphospho-N-acetyl-2-amino-2-deoxyglucose; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Ray, P. H., Kelsey, J. E., Bigham, E. C., Benedict, C. D., Miller, T. A.(1983) in ACS Symposium Series 231 (Anderson, L., and Unger, F. M., eds) pp. 141-169, American Chemical Society, Washington, D. C.
  2. Kohen, A., Berkovich, R., Belakhov, V., and Baasov, T.(1993) Bioorg. Med. Chem. Lett. 3, 1577-1582 [CrossRef]
  3. Dotson, G. D., Nanjappan, P., Reily, M. D., and Woodard, R. W.(1993) Biochemistry 32, 12392-12397 [Medline] [Order article via Infotrieve]
  4. Hedstrom, L., and Abeles, R.(1988) Biochem. Biophys. Res. Commun. 157, 816-820 [Medline] [Order article via Infotrieve]
  5. Tabor, S.(1990) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. eds) pp. Unit 16.2, Green Publishing Associates Inc. and John Wiley & Sons
  6. Kohara, Y., Akiyama, K., and Isono, K.(1987) Cell 50, 495-508 [Medline] [Order article via Infotrieve]
  7. Woisetschlager, M., and Hogenauer, G.(1987) Mol. Gen. Genet. 207, 369-373 [Medline] [Order article via Infotrieve]
  8. Ray, P. H.(1980) J. Bacteriol. 141, 635-644 [Medline] [Order article via Infotrieve]
  9. Lanzetta, P., Alvarez, L. J., Reinach, P. S., and Candia, O. A.(1979) Anal. Biochem. 100, 95-97 [Medline] [Order article via Infotrieve]
  10. Stubbe, J. A., and Kenyon, G. L.(1971) Biochemistry 10, 2669-2677 [Medline] [Order article via Infotrieve]
  11. Stubbe, J. A., and Kenyon, G. L.(1972) Biochemistry 11, 338-345 [Medline] [Order article via Infotrieve]
  12. Clark, V. M., and Kirby, A. J.(1963) J. Am. Chem. Soc. 85, 3705
  13. Bondinell, W. E., Vnek, J., Knowles, P. F., Sprecher, M., and Sprinson, D. B.(1971) J. Biol. Chem. 246, 6191-6196 [Abstract/Free Full Text]
  14. Wooten, E. W., Dua, R. K., Dotson, G. D., and Woodard, R. W.(1994) J. Mag. Res. Ser. A 107, 50-55 [CrossRef]
  15. Banerjee, R. V., Shane, B., McGuire, J. J., and Coward, J. K.(1988) Biochemistry 27, 9062-9070 [Medline] [Order article via Infotrieve]
  16. Metzler, D. E.(1977) in Biochemistry: The Chemical Reactions of Living Cells (Metzler, D. E., ed) pp. 418-419, Academic Press, New York
  17. Anton, D. L., Hedstrom, L., Fish, S. M., and Abeles, R. H.(1983) Biochemistry 22, 5903-5908
  18. DeLeo, A. B., Dayan, J., and Sprinson, D. B.(1973) J. Biol. Chem. 248, 2344-2353 [Abstract/Free Full Text]
  19. Zemell, R. I., and Anwar, R. A.(1975) J. Biol. Chem. 250, 4959-4964 [Abstract]
  20. Hansen, P. E.(1983) Annual Reports of the NMR Spectroscopy (Webb, G. A., eds) Vol. 15, pp. 118-119, Academic Press, London
  21. Hansen, D. E., and Knowles, J. R.(1981) J. Biol. Chem. 256, 5967-5969 [Abstract/Free Full Text]
  22. Hansen, D. E., and Knowles, J. R.(1982) J. Biol. Chem. 257, 14795-14798 [Abstract/Free Full Text]
  23. Kohlbrenner, W. E., Nuss, M. M., and Fesik, S. W.(1987) J. Biol. Chem. 262, 4534-4537 [Abstract/Free Full Text]
  24. Anderson, K. S., and Johnson, K. A.(1990) Chem. Rev. 90, 1131-1149
  25. Marquardt, J. L., Brown, E. D., Walsh, C. T., and Anderson, K. S. (1993) J. Am. Chem. Soc. 115, 10398-10399

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.