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J. Biol. Chem., Vol. 278, Issue 35, 32771-32777, August 29, 2003
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¶
From the
Departments of Medicinal Chemistry and
Chemistry, University of Michigan, Ann Arbor,
Michigan 48109-1065
Received for publication, April 8, 2003 , and in revised form, June 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The synthesis and activation of KDO involves four sequentially acting enzymes: D-arabinose 5-phosphate isomerase (API), deoxy-D-manno-octulosonate 8-phosphate (KDO 8-P) synthase, KDO 8-P phosphatase, and cytidine 5'-monophosphate-KDO synthetase (Fig. 1) (12, 13). The first committed step to KDO generation is catalyzed by 3-deoxy-D-manno-octulosonate 8-phosphate (KDO 8-P) synthase, which condenses D-arabinose 5-phosphate (A5P) with phosphoenolpyruvate via a stereospecific aldol-type condensation (14, 15). A5P is the first intermediate unique to the KDO biosynthetic pathway, and API is the main de novo source of A5P in Gram-negative bacteria, because it is not readily available via glycolysis (16). API synthesizes A5P by catalyzing the reversible aldol-keto isomerization of D-ribulose 5-phosphate (Ru5P) to A5P (Fig. 1). It is reasonable to speculate that inhibition of API will have similar cellular effects as direct KDO synthase inhibition (7–9), thus providing another viable Gram-negative antibiotic target from the KDO pathway.
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The gene(s) encoding API in E. coli K-12 has yet to be positively identified. Recent work in this laboratory provided evidence that the open reading frame (ORF) yrbI encoded a 188-amino acid phosphatase that was highly specific for KDO 8-P (17). Analysis of the E. coli K-12 MG1655 genome (18) at the National Center for Biotechnology Information (NCBI) web site revealed an ORF (yrbH) located next to yrbI in the yrb cluster encoding a putative phosphosugar isomerase. The yrbH gene was cloned, overexpressed, and purified to homogeneity. Utilizing a newly developed 96-well microplate assay, the recombinant protein was subsequently characterized and shown to catalyze the interconversion of A5P and Ru5P. This is the second unknown gene identified from the E. coli yrb cluster to be implicated in KDO biosynthesis.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-glucose
1-phosphate disodium salt and carbazole were from Aldrich. High grade
Spectra/Por® 7 dialysis tubing (10,000 molecular weight cutoff and
metal-free) was obtained from VWR Scientific. The Centriprep YM-10
concentrators and Millex® syringe-driven filter units (0.22
µM) were purchased from Millipore. Hi LoadTM Q-Sepharose
(16/10) fast flow, phenyl Superose (HR 10/10), Superose 12 (HR 10/30), and
fast desalting (HR 10/10) chromatography columns were purchased from Amersham
Biosciences. Polypropylene 96-well full-skirted reaction PCR plates were
obtained from Life Science Products Inc. Corning polystyrene 96-well
flat-bottom assay microplates were from Fisher Scientific. Microplates were
read using the Model 550 microplate reader from Bio-Rad. Sequence Analysis—Data base searching was performed using the BLAST program at the NCBI website (www.ncbi.nlm.nih.gov/BLAST) (19). Clustal W was used to generate multiple sequence alignments (www.ebi.ac.uk/clustalw) (20).
Protein Concentration Assay—Protein concentrations were determined using the Bio-Rad protein assay reagent assay with bovine serum albumin serving as the standard.
Cloning, Overexpression, and Purification of yrbH—Template DNA containing the yrbH gene was prepared by isolating the plasmid from the host cell line. Forward (5'-CATATGTCGCACGTAGAGTTACAACCGGG-3') and reverse (5'-GATTCTGAATTCGGATCCA AGCTTACACTACGCCTGCACGCAGTAAATCATGCATgTGTAACACACCG-3') primers were designed to incorporate NdeI and BamHI sites, respectively. In addition, the reverse primer was designed to remove an endogenous NdeI cut site (lowercase letters). The gene was amplified using standard PCR methodology (FailsafeTM PCR 2x premix buffer "F") and directly ligated into the pCR®T7/CT-TOPO® vector. Chemically competent E. coli TOPO10'F cells were transformed with the recombinant vector, and plasmids were screened by restriction digest. A correct plasmid that had been verified by sequencing to contain the yrbH gene was then digested (NdeI and BamHI) and ligated into a similarly restricted pT7–7 expression vector that had been treated with calf intestinal alkaline phosphatase. The ligation mixture was used to transform chemically competent E. coli XL1-Blue cells, and a transformant harboring the correct pT7-yrbH plasmid was identified by restriction analysis and sequencing. E. coli BL21(DE3) cells were transformed with the plasmid, rechecked by restriction analysis, and stored at –80 °C.
The E. coli BL21(DE3)/pT7-yrbH cells were grown in 2 x YT
medium containing ampicillin (100 mg/liter) at 37 °C with shaking (250
rpm). Once the culture reached the mid-logarithmic growth phase
(A600 
0.7–0.9), the culture was allowed to cool to 18
°C before being induced with isopropyl-
-D-thiogalactoside
at a final concentration of 0.4 mM. After 16 h of growth at 18
°C, the cells were harvested by centrifugation (6,500 x g,
15 min, 4 °C). The cell pellet (3.5 g wet weight) was suspended in 20 ml
of buffer A (20 mM Tris-HCl, pH 8.0) and then sonicated on ice (5
x 30 s; 2-min pauses between pulses). Cellular debris was removed by
centrifugation (29,000 x g, 30 min, 4 °C) and the
supernatant (23 ml; 265 mg of protein) was filtered through a
0.22-µM Millex® filter. The solution was loaded onto a Hi
LoadTM (16/10) Q-Sepharose fast flow column that had been
pre-equilibrated with buffer A. The protein was eluted using a 0–500
mM gradient of NaCl in buffer A over 90 min. Fractions containing
primarily yrbH (35 kDa) as judged by SDS-PAGE were pooled (28 ml; 168 mg
of protein). A saturated solution of AS was slowly added with stirring at room
temperature until 22% saturation was reached (final volume of 38 ml). At this
point, the solution was clarified by centrifugation (29,000 x
g, 30 min, room temperature) and 10 ml was loaded onto a phenyl
Superose (HR 10/10) column that had been equilibrated with a 25% saturated AS
solution of buffer A. A reverse gradient of 25–0% saturated AS solution
in buffer A over 60 min was used to elute the protein, with the majority of
the 35-kDa protein appearing as a single peak after the end of the gradient.
This peak from each of four runs was pooled (35 ml; 112 mg of total protein),
dialyzed against 2 liters of buffer A overnight at 4 °C, and then
concentrated. The preparation was greater than 98% homogeneous as judged
by SDS-PAGE. The final total yield of homogeneous protein was 112 mg/500 ml
cell culture. The purified enzyme was aliquoted (4.7 mg/ml), frozen in
ethanol-dry ice, and stored at – 80 °C.
One-dimensional Polyacrylamide Gel Electrophoresis—SDS-PAGE
was performed on protein samples (5–10 µg) under reducing
conditions on a 12% polyacrylamide gel with a Mini-PROTEAN II electrophoresis
unit (Bio-Rad). Gels were stained with 0.25% Coomassie Brilliant Blue R250
solutions.
Molecular Weight (MW) Determinations—The subunit MW of yrbH was determined by matrix-assisted laser desorption ionization mass spectrometry on a VESTEC-2000 instrument using a sinipinic acid matrix by the University of Michigan Protein Structure Facility. Protein samples were prepared by buffer exchange via overnight dialysis against 2 liters of 5 mM HEPES, pH 7.5, at 4 °C. The native MW was determined by gel filtration utilizing a Superose 12 column (HR10/30) with 150 mM NaCl in buffer A as eluent. The elution volume was determined in triplicate for all samples and standards.
API Activity Assay—The API activity was determined by a 96-well microplate adaptation of the discontinuous cysteine-carbazole colorimetric assay initially developed by Dische and Borenfreund (21) and later modified by Ray and coworkers (22). A solution containing 25 µlof enzyme at various concentrations in buffer (100 mM Tris-HCl, pH 8.5) was incubated at 37 °C for 3 min in a 96-well reaction PCR plate using a Peltier thermal cycler. The reaction was initiated with 25 µl of a 2x mM A5P solution and subsequently quenched after various time intervals with 50 µl of 25 N H2SO4. A 90-µl aliquot was transferred to a flatbottom assay plate containing 250 µl of a freshly prepared H2SO4-cysteine-carbazole mixture (230 µl of 25 N H2SO4: 10 µl of an aqueous cysteine 1.5% (w/v) solution: 10 µl of a 0.12% (w/v) ethanolic carbazole solution) in each well. Plates were thoroughly mixed, and the color was allowed to develop at room temperature for 1 or 3 h, depending on the assay. It was observed that the sensitivity for ketose detection and the degree of reproducibility were increased by longer color development times at room temperature (data not shown) as opposed to shorter development times with heating (22). However, longer development times reduced the linear range, presumably because of the instability of the chromophore. Accordingly, plates being monitored for the appearance of ketose were read at 540 nm after 3 h of color development, and the plates being monitored for the disappearance of ketose were read after 1 h. All plates contained internal Ru5P standards and appropriate A5P controls in triplicate. One unit of enzyme activity is defined as the conversion of 1 µmol of sugar phosphate per minute at 37 °C.
Metal Content Analysis—Enzyme samples as isolated above were prepared for metal analysis by extensive dialysis (48 h) against 2 liters of metal-free buffer B (20 mM Trizma-HCl, pH 8.5) at 4 °C. Metal-free protein samples were prepared by first incubating enzyme in the presence of 10 mM EDTA for 2 h at 4 °C, followed by isolation using a fast desalting column (HR 10/10) with buffer B as eluent. Divalent metal content of the as-isolated and EDTA-treated samples were determined by high-resolution inductively coupled plasma-mass spectrometry on a Finnigan MAT ELEMENT instrument at the University of Michigan Department of Geology.
Effect of Divalent Metals on Activity—Enzyme samples of yrbH as isolated were diluted with buffer C (100 mM Trizma-HCl, pH 8.5) and incubated with various divalent metals or EDTA for 30 min at 4 °C. Activity was assayed at 37 °C under saturating substrate conditions in triplicate with a 3-min reaction time and 3-h color development (final API, 70 nM; A5P, 10 mM; metal or EDTA, 10 µM). EDTA-treated samples were desalted as described and incubated with various concentrations of Zn2+ at 4 °C for 30 min. The remaining activity was measured in triplicate at 37 °C with a 2-min reaction and 3-h color development (final API, 15 nM; A5P, 10 mM; Zn2+, 0–25 µM).
Optimum pH and pKa Determination—The activities
of yrbH in solutions of varying pH values (6.25–10) were measured by
diluting the enzyme in 200 mM BTP buffers containing 2
mM EDTA. The pH of the buffers was adjusted at 37 °C. Activity
was measured as outlined above in triplicate with a 3-min reaction time and
3-h color development (final API, 70 nM; A5P, 10 mM;
EDTA, 1 mM). The bell-shaped Vapp data were fit
to Equation 1 using nonlinear
least-squares fitting to estimate pKa values.
 | (Eq. 1) |
Substrate Specificity—Enzyme samples were diluted in buffer C containing 2 mM EDTA and assayed by initiating the reaction with 25 µl of 20 mM sugar (final API, 70 nM; EDTA, 1 mM; sugar, 10 mM). After 10 min at 37 °C, reactions containing the potential alternate substrates D-arabinose, D-ribose 5-phosphate, D-glucose 6-phosphate, D-glucose 1-phosphate, D-glucosamine 6-phosphate, or D-mannose 6-phosphate were quenched and the presence of ketose was determined as outlined above for A5P. Each carbohydrate was assayed in triplicate, along with appropriate controls from which the addition of enzyme was withheld. The lower limit of detection for this assay is estimated to be under 1% of ketose formation. Because the respective ketose forms of D/L-glyceraldehyde 3-phosphate and D/L-erythrose 4-phosphate do not readily form chromophores under the assay conditions used, product appearance was monitored by 31P NMR for these two substrates.
Determination of Kinetic Parameters—Reactions were performed at 37 °C using the discontinuous microplate assay as outlined above, using buffer C containing 2 mM EDTA to dilute the enzyme stock solution. Substrate concentrations typically ranged from a Km of 0.2 to 10. After 2 min the reactions (final API, 7 nM; EDTA, 1 mM) were quenched, at which point approximately less than 10% of substrate had been consumed. Color was developed for 1 or 3 h when using Ru5P or A5P as substrate, respectively. Initial rates (v0) were determined in triplicate and fit to the standard Michaelis-Menten equation using nonlinear least-squares regression to determine values for Km and kcat.
Equilibrium (Keq) Determination—Solutions containing either A5P or Ru5P (0.5, 1, or 2 mM) were incubated in buffer C with 1 mM EDTA at 37 °Cfor4hinthe presence of enzyme (API, 700 nM). Reactions were quenched and color developed as above (1 h). In a separate experiment, solutions containing either 5 mM A5P or 5 mM Ru5P in 50 mM Trizma-HCl, pH = 7.5, with 1 mM EDTA and 10% D2O were incubated in the presence of 700 nM API at 37 °C. Reactions were periodically monitored by 31P NMR using a Bruker Avance DRX-300 instrument with WALTZ16 proton decoupling. A 10-s delay (>3 times the T1A5P/Ru5P) was used during the acquisition to ensure complete relaxation of the phosphorus nucleus, thus allowing direct integration and comparison of peak areas for the two different sugar phosphates under observation. Chemical shifts were referenced to an external phosphoric acid standard (0 ppm). Once there was no change in peak ratios for both samples, the spectra were acquired (64 scans). Keq is reported for API in the direction of Ru5P product formation from A5P substrate (Ru5P/A5P).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Overexpression and Purification of yrbH—The recombinant protein yrbH was overexpressed using the pT7–7 expression vector (Fig. 3). Optimal conditions for the maximal ratio of recombinant to cellular protein were achieved by overnight expression at 18 °C. The protein exhibited a strong propensity to aggregate, particularly at pH values below 7.2 (data not shown). Efficient purification to homogeneity was achieved in two steps using a Q-Sepharose anion exchange column followed by a phenyl Superose column (Table II). The specific activity after dialysis was 246 units/mg. Purified API was stable for at least 1 month when stored at –80 °C.
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Molecular Weight Determination—The subunit molecular weight
of yrbH as determined by SDS-PAGE electrophoresis under reducing conditions
was 35,000 (MWcalc = 35,200)
(Fig. 3). The observed doublet,
which was at first attributed to proteolytic degradation, could not be
prevented by the inclusion of standard protease inhibitors
(phenylmethylsulfonyl fluoride, EDTA, pepstatin) in the lysis buffer. However,
matrix-assisted laser desorption ionization-mass spectrometry indicated the
presence of a single protein species (M+1 = 35,084,
M+2 = 17,540), suggesting the observed doublet in
SDS-PAGE is an artifact of sample degradation during preparation and/or
partial denaturation. The native molecular mass as determined by gel
filtration was 122 ± 5 kDa, indicating API is likely a tetramer
(3.5 subunits).
Metal Content—The metal content of API as isolated was 1.0 ± 0.1 equivalents of Zn2+/subunit as determined by high-resolution inductively coupled plasma-mass spectrometry. Samples that had been treated with EDTA and subsequently isolated by gel filtration contained only trace amounts of metals, including Zn2+ (<0.01 equivalents per monomer), indicating EDTA is an effective chelator of API-bound metal cations.
Effect of Divalent Metals on Activity—The addition of EDTA to API as isolated, without removal of either EDTA-metal complex or excess free EDTA, increased the observed activity nearly 2-fold (Fig. 4A). The Group 2A metals (Mg2+, Ca2+, Ba2+) and the mid-transition metals (Mn2+,Fe2+,Co2+) had no appreciable effect on activity. The late-transition metals (Ni2+, Cu2+,Zn2+,Cd2+,Hg2+) inhibited API, particularly those with the d10 electron configuration. Concentrations of 10 µM Zn2+, Cd2+, or Hg2+ were sufficient to cause complete inhibition. Using enzyme solutions from which all metal had previously been removed by EDTA treatment followed by gel filtration, the IC50 for Zn2+ was estimated between 1–3 µM (Fig. 4B).
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Optimum pH and pKa—A plot of Vapp versus pH data produced a bell-shaped activity profile (Fig. 5). A satisfactory fit (r = 0.98) of the data was achieved using the model as defined by Equation 1. The pH optimum was determined to be 8.4. The presence of an acidic catalytic residue with pKa1 = 6.55 ± 0.04 and a basic catalytic residue with a pKa2 = 10.34 ± 0.07 was implicated in the active site of API.
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Substrate Specificity—The short chain phosphorylated aldoses D/L-glyceraldehyde 3-phosphate and D-erythrose 4-phosphate did not serve as alternate substrates for A5P as determined by 31P NMR (data not shown). In addition, none of the phosphorylated pentoses and hexoses tested (D-ribose 5-phosphate, D-glucose 6-phosphate, D-glucose 1-phosphate, D-glucosamine 6-phosphate, or D-mannose 6-phosphate) were converted to their respective ketoses as monitored by an increase in the ratio of absorbance at 540 nm of standard to control (Table III). D-Arabinose was not a substrate. API thus appears to be a specific phosphosugar aldol-keto isomerase for A5P within the limits of detection of the assay.
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Kinetic Parameters—API follows standard Michaelis-Menten kinetics, and the parameters were determined for both A5P and Ru5P. API has an apparent Km of 0.61 ± 0.06 mM for A5P and 0.35 ± 0.08 mM for Ru5P. The apparent kcat in the A5P to Ru5P direction is 157 ± 4 s–1 and 255 ± 16 s–1 in the Ru5P to A5P direction. The Keq, as determined by the end point assay and confirmed by 31P NMR (Fig. 6), is 0.50 ± 0.06.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Domain analysis of API indicated a core N-terminal SIS (sugar
isomerase) domain (210 amino acids) followed by a pair of
C-terminal CBS (cystathionine
-synthase) domains (50–60 amino acids per
domain) (Fig. 2). SIS domains
are commonly found motifs in aldol-keto isomerases and in proteins that
regulate the expression of genes involved in the synthesis of phosphosugars
(27). CBS domains are found in
numerous proteins; their exact function is currently unknown, although it is
believed that they may play a regulatory role and are implicated in numerous
disease states (28). API is a
logical control point because it catalyzes the first step in the KDO pathway
(Fig. 1) via consumption of
Ru5P, a critical intracellular metabolite common to multiple pathways
(16,
29). Whether there is
CBS-mediated modulation of API activity in order to regulate the flux through
the KDO pathway is yet to be determined.
The characterization of yrbH reported here is consistent with its identification as a specific A5P isomerase in E. coli K-12. The high specific activity (246 units/mg), competent kinetic parameters for both A5P (Km = 0.61 ± 0.06 mM; kcat = 157 ± 4 s–1) and Ru5P (Km = 0.35 ± 0.08 mM; kcat = 255 ± 16 s–1), and substrate specificity (Table III) support the conclusion that yrbH is an A5P isomerase. The equilibrium at 37 °C lies well in favor of A5P (68 ± 2% to 32 ± 2%) (Fig. 6), and the pH optimum is 8.4 (Fig. 5). This value is in agreement, within error, with the Keq as calculated by the Haldane relationship (0.35). The pKa values (6.55 ± 0.04 and 10.34 ± 0.07) suggest the presence of a histidine or possibly a carboxylate along with a lysine or arginine at the catalytic site. It is unlikely that the later pKa is due to a cysteine because DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)0 titrations (30) detected only one modifiable cysteine that could not be protected by co-incubation with substrate (data not shown). These same amino acids are evoked as general base/acid catalytic residues in two other well studied isomerases, glucose 6-phosphate isomerase (31) and triose phosphate isomerase (32, 33), and may reflect a common mechanism. Recombinant API contained approximately one zinc atom per monomer after purification. The metal could be removed by treatment with EDTA followed by gel filtration and resulted in nearly a 2-fold increase in observed activity. In addition, the activity was increased upon the in situ addition of excess EDTA (Fig. 4A), and thus there does not appear to be a metal cofactor requirement. API is extremely sensitive to d10 metal inhibition (Fig. 4A), with an estimated IC50 = 1–3 µM for zinc (Fig. 4B). This reflects the upper limit, and the true inhibition constant is likely much lower, particularly if ZnOH+ is the inhibitory species. Submicromolar reversible zinc inhibition has been observed in other enzymes (34), including phosphomannose isomerase (35). The mode of inhibition in API may be through coordination to active site carboxylate and/or histidine residues, which are commonly encountered in high affinity zinc ligands (36, 37). It has been speculated that metal ion inhibition may be a mechanism to control activity in vivo (34), although the biological significance in terms of API is unknown.
The retention of two seemingly paralogous genes in E. coli K-12 (yrbH, gutQ) and three in the uropathogenic E. coli CFT073 (yrbH, gutQ, kpsF) genome is of considerable interest (Fig. 2). Based on homology studies, yrbH appears to be the most ubiquitously conserved among Gram-negative bacteria (Table I). The kpsF and gutQ genes, however, are not as widely distributed. The kpsF gene appears to be confined to the first ORF of region 1 of the kps locus in E. coli strains expressing Group 2 type capsules (38, 39) and is likely involved in A5P (i.e., KDO) production for capsule biosynthesis. The function of gutQ, which is located in the glucitol operon (40), is currently unknown. The domain architectures of kpsF and gutQ are similar to yrbH except in the N-terminal segments (Fig. 3). The significance of the sequence variation is not clear.
In summary, the yrbH gene has been shown to encode for a protein containing API activity. This is the second unknown ORF identified from the yrb cluster. The protein was cloned, overexpressed, and characterized. Work is currently in progress to characterize the other E. coli proteins (gutQ and kpsF) with homology to yrbH in order to confirm their function in vitro, to explore the possibility of regulation at the post-translational level, and to gain further insight into the mechanism of coordination of API(s) in Gram-negative bacteria.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: College of Pharmacy, 428 Church St., Ann Arbor, MI 48109-1065. Tel.: 734-764-7366; Fax: 734-763-2022; E-mail: rww{at}umich.edu.
1 The abbreviations used are: LPS, lipopolysaccharide; KDO,
3-deoxy-D-manno-octulosonate; KDO 8-P,
3-deoxy-D-manno-octulosonate 8-phosphate; API,
D-arabinose 5-phosphate isomerase; A5P, D-arabinose
5-phosphate; Ru5P, D-ribulose 5-phosphate; AS, ammonium sulfate;
ORF, open reading frame; MW, molecular weight.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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