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Plant Physiol. (1998) 118: 199-207
D-Ribulose-5-Phosphate 3-Epimerase: Cloning and
Heterologous Expression of the Spinach Gene, and Purification and
Characterization of the
Recombinant Enzyme1
Yuh-Ru Chen,
Fred C. Hartman*,
Tse-Yuan S. Lu, and
Frank W. Larimer
University of Tennessee-Oak Ridge Graduate School of Biomedical
Sciences, Oak Ridge, Tennessee 37831 (Y.-R.C.); and Protein
Engineering Program, Life Sciences Division, Oak Ridge National
Laboratory, Oak Ridge, Tennessee 37831 (F.C.H., T.-Y.S.L., F.W.L.)
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ABSTRACT |
We have achieved, to our knowledge,
the first high-level heterologous expression of the gene encoding
D-ribulose-5-phosphate 3-epimerase from any source, thereby
permitting isolation and characterization of the epimerase as found in
photosynthetic organisms. The extremely labile recombinant spinach
(Spinacia oleracea L.) enzyme was stabilized by
DL- -glycerophosphate or ethanol and destabilized by
D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this
lability, the unprecedentedly high specific activity of the purified
material indicates that the structural integrity of the enzyme is
maintained throughout isolation. Ethylenediaminetetraacetate and
divalent metal cations did not affect epimerase activity, thereby
excluding a requirement for the latter in catalysis. As deduced from
the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to
these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.
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INTRODUCTION |
As a participant in the oxidative pentose phosphate pathway, Ru5P
epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P and
Xu5P, is widely distributed throughout nature. Beyond its catabolic
role, the epimerase is also vital anabolically to photosynthetic
organisms in the regenerative phase of the reductive pentose phosphate
pathway (the Calvin cycle). In this capacity, Ru5P epimerase directs
Xu5P, formed in two distinct transketolase reactions of the cycle, to
Ru5P. Phosphorylation of the latter regenerates
D-ribulose-1,5-bisphosphate, the substrate for net CO2 fixation. Because both the oxidative and
reductive pentose phosphate pathways coexist in chloroplasts
(Schnarrenberger et al., 1995 ), Ru5P epimerase and R5P isomerase
facilitate partitioning of pentose phosphates between the two pathways,
as dictated by the metabolic needs and redox status of the cell.
Scant structural and mechanistic information about Ru5P epimerase is
available despite its inherent importance and dual metabolic roles.
This neglect may in part reflect the low natural abundance of the
enzyme. For example, achievement of electrophoretic homogeneity required a 2000-fold purification from yeast (Bär et al., 1996) and spinach (Spinacia oleracea L.) chloroplasts (Teige et
al., 1998) and 9000-fold purification from beef liver (Terada et al., 1985). Although low overall recoveries (<10%) further limited the
availability of pure material, molecular sieving and denaturing electrophoresis established that the epimerases from mammals (Wood, 1979; Karmali et al., 1983; Terada et al., 1985) and yeast (Bär et al., 1996) are homodimers of approximately 23-kD subunits, whereas
the enzyme from spinach chloroplasts may be an octamer of 23-kD
subunits (Teige et al., 1998). DNA-deduced amino acid sequences of Ru5P
epimerases from both photosynthetic and nonphotosynthetic sources,
which confirm this estimated subunit size, show greater than 50%
similarities among the most evolutionarily distant species examined
(Kusian et al., 1992; Blattner et al., 1993; Falcone and Tabita, 1993;
Lyngstadaas et al., 1995; Nowitzki et al., 1995; Teige et al.,
1995).
Although Ru5P epimerase has very recently been purified from a
photosynthetic organism (spinach) for the first time (Teige et al.,
1998), the low recovery (100 µg from 3.8 g of soluble chloroplast protein, representing an overall yield of 5%) imposes severe constraints on the directions of future experiments.
Furthermore, despite successful cloning of cDNA fragments encoding Ru5P
epimerase of several photosynthetic organisms (Kusian et al., 1992;
Nowitzki et al., 1995; Teige et al., 1995), to our knowledge high-level heterologous expression and purification of enzymically active recombinant enzyme have not been achieved. Because of our interest in
the regulation of photosynthetic carbon assimilation and the requisite
need for ample supplies of the participant enzymes for use in
mechanistic studies, we have attempted to optimize the heterologous
expression of the spinach gene for Ru5P epimerase. In this paper we
report cDNA clones that encode the mature chloroplastic enzyme or its
cytoplasmic precursor. We also describe an efficient isolation
procedure for the mature spinach enzyme synthesized in
Escherichia coli and some of the properties of the purified enzyme. Contrasting features of the plant Ru5P epimerase, relative to
the animal and yeast counterparts, include an octameric rather than a
dimeric structure (also see Teige et al., 1998) and striking instability under routine laboratory conditions.
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MATERIALS AND METHODS |
Materials
Materials and vendors were as follows: a library of spinach
(Spinacia oleracea L. cv Melody) cDNAs in  ZAPII and
Pfu DNA polymerase to use for PCR, Stratagene;
[35S]dATP, New England Nuclear; T4 DNA ligase,
New England Biolabs; nitrocellulose filters (82 mm) for plaque
hybridization, Schleicher & Schuell; transketolase, triose phosphate
isomerase, glycerophosphate dehydrogenase, thiamine pyrophosphate, G3P,
R5P, Ru5P, leupeptin, and PMSF, Sigma; 4-(2-aminoethyl)benzenesulfonyl
fluoride, Calbiochem; and 3,3 -diaminobenzidine tetrahydrochloride
dihydrate, Bio-Rad. Common laboratory reagents for enzyme purification
and assays were procured at the highest level of purity readily
available. Phosphoribulokinase was prepared as described previously
(Porter et al., 1986, 1988).
Rabbit serum containing polyclonal antibodies raised against purified
recombinant spinach epimerase (a mixture of peaks I and II, see below)
was prepared by Berkeley Antibody Company (Richmond, CA).
Cloning and Heterologous Expression of rpe, the Spinach
Gene for Ru5P Epimerase
Oligonucleotide primers for PCR, mutagenesis, and dye-terminator
sequencing were prepared with a PCR-Mate (Applied Biosystems) based on
phosphoramidite chemistry. Synthesized PCR primers duplicated sequences
of the potato rpe cDNA (Teige et al.,
1995)2 that encode
two highly conserved regions of the epimerase: PSILSANF (residues
17-24)3 and MSVNPGFG
(residues 149-156)3 (see Nowitzki et al., 1995; Teige et
al., 1995). These primers (ccg tcc atc ctt tct gct aac tt for the
coding strand and cca aat cca ggg ttt aca gac at for the complementary
strand) were used to amplify the 419-bp fragment, which brackets the
two conserved regions, from an aliquot of the spinach cDNA library that
contained 106 plaque-forming units. The PCR
product was cloned into an SmaI site of plasmid
pBS+ (Stratagene), sequenced to confirm anticipated
homology to the potato rpe, and reamplified from the plasmid
as a template. Subsequently, [35S]dATP was
introduced into the reamplified PCR product with a random priming
kit (United States Biochemical).
The labeled PCR product served as a probe in plaque-hybridization
screens of the spinach cDNA library according to the manufacturer's protocol. Six positive clones were obtained from the 3 × 104 plaque-forming units screened. Two of these
encompassed the entire coding sequence, inclusive of the N-terminal
transit appendage, for spinach Ru5P epimerase, as shown by complete
sequencing of both strands.
As an initial step in the construction of expression vectors, the 3
untranslated region of the cDNA was truncated at the BamHI
site. For generation of the transit form of the epimerase, an
NcoI site was introduced by site-directed mutagenesis
(Kunkel et al., 1987) at the Met initiation codon, coincidentally
replacing the adjacent codon for Ser with a codon for Gly. For
generation of the mature form of the epimerase, an NcoI site
was introduced at the codon for Lys-48 of the transit protein,
resulting in conversion to a Met initiation codon. These engineered
clones were individually ligated as NcoI-BamHI
fragments adjacent to the tac promoter of the expression vector pFL260
(Larimer et al., 1990) (Fig. 1). The
ligated fragments of the expression vectors were sequenced to confirm
the desired structures.
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| Figure 1.
Sequence of expression cassettes for spinach
recombinant Ru5P epimerase. A, Transit protein expression cassette
(accession no. AF070942). The tac promoter, operator, and
ribosome-binding site (rbs) are shown in lowercase; the coding sequence
is in uppercase. B, Mature epimerase expression cassette (accession no.
AF070943).
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An overnight culture (25 mL) of the appropriate expression vector in
host strain MV1190 or XL 1-Blue, grown in 2× YT medium (Sambrook et
al., 1989) containing 1% (v/v) glycerol and 50 µg mL 1 ampicillin at 37°C, was diluted 1:100
into the same medium and incubated for 4 h with vigorous
shaking (250 rpm). Isopropyl  -D-thiogalactopyranoside was added to 0.1 mM, and the incubation was continued for
an additional 3 h at 37°C followed by harvesting of the cells by
centrifugation.
Purification of Recombinant Ru5P Epimerase
All steps of the purification were carried out at 2°C to 4°C,
and all buffers were at pH 8.0. When G3P was the only buffer component,
solutions of the commercial disodium salt were adjusted to pH 8.0 with
0.1 N HCl. Selection of fractions to pool from chromatographic steps was based on both epimerase assays and SDS-PAGE. Cell paste (approximately 15 g) from 1 L of culture was
suspended in a buffer containing 50 mM Bicine, 10 mM G3P, 1 mM EDTA, 1 mM DTT, 10 µM leupeptin, 200 µM
4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM PMSF, and
5% (v/v) glycerol. The slurry was passed twice through a French
pressure cell at 12,000 to 16,000 p.s.i. and ultracentrifuged at
100,000g for 1 h. The supernatant was adjusted to pH
8.0 with 1 M Tris (free base form), diluted 1.5-fold with water, and applied to a DE52 anion-exchange column (2.5 × 10 cm, Whatman) that had been equilibrated with 10 mM G3P. After
the column was washed with 200 mL (about 4 column volumes) of 10 mM G3P to remove all unbound materials, elution was
continued with 10 mM G3P containing 50 mM NaCl.
Epimerase emerged as a rather sharp peak just beyond the breakthrough
volume. Pooled fractions were concentrated (Centriprep-30, Amicon,
Beverly, MA) to about 8 mL, adjusted to 5 mM potassium
phosphate by the addition of 1% (v/v) 10 mM G3P/500
mM potassium phosphate, and filtered (0.45 µm, Gelman
Acrodisc, Ann Arbor, MI). The filtered sample was applied to a 1.6- × 10-cm column of hydroxyapatite (ceramic hydroxyapatite type I, particle
size of 40 µm, Bio-Rad), adapted to a fast-protein liquid
chromatography unit (Pharmacia LKB Biotech Inc.), which had been
equilibrated with 10 mM G3P/5 mM potassium
phosphate. Upon isocratic elution of the column with equilibration
buffer, epimerase emerged essentially with the solvent front. Peak
fractions were pooled, concentrated (Centriprep-30) to about 8 mL, and
dialyzed against 10 mM G3P/1 mM EDTA.
Subsequent to dialysis, the sample was filtered (0.45 µm) and applied
to a prepacked Mono-Q column (HR10/10), adapted to a fast-protein
liquid chromatography unit, which had been equilibrated with 10 mM G3P/1 mM EDTA. Elution of the column with an
80-mL linear gradient of 0 to 100 mM NaCl in equilibration
buffer resolved two major peaks of protein (Fig. 2) that contained epimerase activity. The
major peak (75% of the total activity recovered) emerged at 40 mM NaCl, whereas the minor peak appeared at 65 mM NaCl; both peaks of epimerase had the same specific
activity. Fractions from the two peaks were pooled separately, concentrated to >2 mg mL1, and stored
frozen at  80°C. A summary of the purification protocol is provided
in Table I.
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| Figure 2.
Resolution of two peaks of epimerase by
anion-exchange chromatography on Mono-Q. See text for additional
details.
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Ru5P Epimerase from Spinach
About 1 g of fresh spinach leaves was ground with a mortar
and pestle in 1 mL of extraction buffer as used for Escherichia coli, and the resulting slurry was centrifuged at 4°C for 20 min at 4600g. The Ru5P epimerase in the crude supernatant was
assessed by western-blot analyses of polyacrylamide gel
electropherograms.
Protein and Enzyme Assays
Protein concentration was determined with Bradford's reagent
(Bradford, 1976) with BSA as the standard. Ru5P epimerase activity was
assayed spectrophotometrically at 340 nm and 25°C (Kiely et al.,
1973). Assay solutions (120 µL) at pH 8.0 routinely contained 50 mM Bicine, 1 mM EDTA, 0.1 mM
thiamine pyrophosphate, 0.25 mM NADH, 5 mM R5P,
1 mM Ru5P, 0.12 unit of transketolase, 2.4 units of triose
phosphate isomerase, 0.24 unit of glycerophosphate dehydrogenase, and
1.6 × 103 to 2.4 × 103 units of Ru5P epimerase. Before the
addition of the epimerase, the assay solution was preincubated for 5 min to ensure complete consumption of the Xu5P that was present as a
contaminant in the commercial Ru5P preparation. One unit of epimerase
activity is defined as the oxidation of 1 µmol NADH
min1 in the 120-µL assay solution.
In kinetic studies for Vmax,
Km, and Ki
determinations, the concentration of R5P in assay solutions was
decreased from 5 to 2 mM, a change that did not diminish
the measured epimerase activity. Use of a lower concentration of R5P
was prompted by the finding that the commercial preparation contained
2% contamination by Ru5P, as estimated by analysis with
phosphoribulokinase (Porter et al., 1986). Hence, at 5 mM
R5P, Ru5P is concurrently introduced at 0.1 mM, a
concentration that exceeds the lowest needed for reliable determination
of its Km.
Molecular Mass Estimations
The subunit molecular mass of Ru5P epimerase was determined by
SDS-PAGE at 15°C on 12.5% (w/v) PhastGels in conjunction with a
PhastSystem apparatus (Pharmacia Biotech). Standards were provided by
the low-molecular-mass calibration kit from Pharmacia Biotech. Gels
were stained with silver or Coomassie blue according to the supplier's
protocol.
The molecular mass of native Ru5P epimerase was estimated by gel
filtration and by PAGE under nondenaturing conditions on gradient gels.
A prepacked Superose 12 HR column (1.0 × 30 cm; Pharmacia
Biotech) was equilibrated with a pH 8.0 buffer (10 mM G3P,
150 mM NaCl, and 1 mM EDTA) and calibrated with
a gel-filtration calibration kit (inclusive of proteins from 25 to 440 kD) from the same vendor. The elution position of the epimerase (0.2 mg in 0.1 mL of equilibration buffer applied to the column) was then determined and assessed relative to the calibration profile.
Nondenaturing PAGE was carried out at 15°C on 8% to 25% (w/v)
gradient PhastGels; correlation of electrophoretic mobility with
protein size was provided by the high-molecular-mass calibration kit
from Pharmacia Biotech. Gels were fixed with TCA and stained with
Coomassie blue according to the manufacturer's instructions.
pI of Ru5P Epimerase
IEF on polyacrylamide gels was used to assess the purity of
epimerase preparations and also to determine the pI of the epimerase subunit under denaturing conditions and the pI of the holoenzyme under
nondenaturing conditions. For the latter, PhastGels IEF 4 to 6.5 and
the broad pI calibration kit were used according to the manufacturer's
protocol, except that the temperature during focusing was maintained at
5°C to mitigate denaturation of the epimerase. IEF under denaturing
conditions at 25°C was achieved with PhastGels IEF 5 to 8 that were
presoaked for 10 min in 2% (v/v) ampholyte (pH 5.0-8.0) containing
9.5 M urea, quickly rinsed by dipping into water, and
gently blotted to remove excess liquid before sample application. The
epimerase sample was diluted with 2 volumes of 9.5 M urea
containing 5% (v/v) 2-mercaptoethanol. Focused gels were stained with
Coomassie blue in accordance with the manufacturer's instructions.
Equilibrium Constant
The equilibrium ratio of Ru5P to Xu5P, as established by the
epimerase at 25°C and pH 8.0, was determined by the incubation of 2 mM Ru5P with either 0.03 or 3 µg of the purified enzyme
in 1.2 mL of 50 mM Bicine and 1 mM EDTA.
Periodically, aliquots (400 µL) were removed, deproteinated by
centrifugation in Centricon-10 tubes (Amicon), and assayed for Ru5P
with phosphoribulokinase (Porter et al., 1986) and for Xu5P with
transketolase (the same assay solution used for epimerase activity but
lacking the epimerase). The epimerase reactions were monitored until
the concentrations of Ru5P and Xu5P were constant.
Western Blots
Ru5P epimerase was detected on polyacrylamide gels by western
immunoblotting (Towbin et al., 1979; Burnette, 1981; Jahn et al., 1984;
Johnson et al., 1984). Electrophoresis of samples under both denaturing
and nondenaturing conditions and IEF under nondenaturing conditions
relied on an XCell Mini-Cell apparatus (Novex, San Diego, CA) with
gels, buffers, and prestained molecular-mass markers supplied by the
same vendor. Denaturing electrophoresis at room temperature was
achieved on 4% to 12% (w/v) NuPAGE Tris gels with Mes-SDS running
buffer, and nondenaturing electrophoresis at 2°C was achieved on 8%
to 16% (w/v) Tris-Gly gels with Tris-Gly native running buffer. IEF at
2°C entailed the use of pH 3.0 to 7.0 IEF gels with associated
cathode and anode buffers. Proteins from nonstained gels were
transferred to nitrocellulose membranes with an XCell Mini-Cell and
Blot Module (Novex). The membranes were soaked for 10 min in 25% (v/v)
isopropanol/10% (v/v) acetic acid, rinsed with water, blocked for 30 min with 5% (w/v) nonfat dry milk dissolved in pH 7.4 buffer (50 mM Tris, 200 mM NaCl), and placed in a
1:3000-fold dilution (with Tris buffer inclusive of 0.1% [v/v]
Triton X-100) of the serum containing epimerase antibodies. After a 1-h
incubation, the membranes were rinsed several times with the
Tris-Triton X-100 buffer and then immersed for 30 min in
peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad), which was diluted
3000-fold with the Tris-Triton X-100 buffer. Color was developed in the
Tris buffer containing 1.3 mM 3,3-diaminobenzidine tetrahydrochloride dihydrate and 9 mM
H2O2.
Sequencing
DNA sequencing was carried out on a sequencer (model 373A, Applied
Biosystems) by use of either dye-primer or dye-terminator chemistries.
Edman degradation of SDS-denatured Ru5P epimerase (a mixture of peaks I
and II; 1 nmol) was accomplished with a gas-phase sequencer (model 470, Applied Biosystems) equipped with a 120A analyzer and 900A control
module.
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RESULTS |
Cloning and Expression of rpe Spinach cDNA
Our clones of the rpe cDNA (accession no. AF070941)
differ from that reported by Nowitzki et al. (1995) and the
corresponding accession (no. L42328) only in the 5 and 3 untranslated
regions, which may reflect the construction of cDNA libraries from
different cultivars of spinach (cv Melody versus cv Monnopa). Our
clones are longer on both ends compared with L42328; because we observe the same 5 end in two independent clones, it may represent the true 5
end of the mRNA without the cap. Poly(A+) tracts were not
observed on the 3 ends of any of the cDNA clones. In addition to
construction of an expression vector for the mature form of Ru5P
epimerase (Fig. 1B), we also designed one for the transit form of the
enzyme (Fig. 1A), in anticipation of chloroplast import studies.
However, based on western-blot analyses of crude extracts, E. coli completely processed the transit protein to mature epimerase
(data not shown). Not surprisingly, epimerase activity levels in crude
extracts of E. coli, whether transformed with the plasmid
encoding the transit or the mature form of the enzyme, were found to be
about the same. Thus, we have not addressed the question of intrinsic
activity of the transit protein.
Assessment of Purity, Size, and Charge of Ru5P Epimerase
The isolation scheme summarized in Table
I provides highly purified recombinant
enzyme in reasonable overall recovery (35%). Based on purification
level, the epimerase represents about 2% of the total soluble proteins
extracted from the host. Beginning with the centrifuged crude extract
from transformed E. coli, progression of the purification
was readily monitored by SDS-PAGE (Fig.
3A). The DE52 column was particularly
effective in rendering enrichment of the epimerase, which was only
weakly adsorbed at pH 8.0 relative to most other components in the
crude extract. Upon passage of the preparation through hydroxyapatite,
the epimerase emerged with only minor contaminants and will thus be
suitable for a variety of studies. Although these contaminants were
effectively removed by Mono-Q chromatography, which resolved two peaks
(I and II) of epimerase, the specific activity was somewhat compromised
because of a modest loss of units during the preceding dialysis.
Neither their specific activities (Table I) nor their mobilities during SDS-PAGE (Fig. 3B) distinguished the two peaks.
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| Figure 3.
Progression of purification of spinach recombinant
epimerase as assessed by SDS-PAGE. A, Gel stained with Coomassie blue
includes: lane 1, molecular mass markers (bands from top to
bottom: phosphorylase b, 94 kD; albumin, 67 kD;
ovalbumin, 43 kD; carbonic anhydrase, 30 kD; trypsin inhibitor, 20.1 kD; lactalbumin, 14.4 kD); lane 2, centrifuged extract (3 µg); lane
3, pool from DE52 (2 µg); and lane 4, pool from hydroxyapatite (0.5 µg). B, Gel stained with silver for increased sensitivity includes:
lane 1, peak I from Mono-Q (20 ng); and lane 2, peak II from Mono-Q (20 ng).
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The subunit size of the epimerase as estimated by SDS-PAGE (Fig. 3B)
was 25,000 D, in close agreement with the 25,085 D tabulated from the
DNA-deduced amino acid composition. Both peaks of epimerase were also
identical and homogeneous, as judged by nondenaturing PAGE, and
essentially co-migrated with beef liver catalase of 232,000 D (Fig.
4). Further examination by gel
filtration, in parallel with appropriate standards, was consistent with
a molecular mass of 200,000 D (data not shown). Taken together, these
data demonstrate that the native epimerase is octameric.
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| Figure 4.
Purity and molecular mass of recombinant epimerase
as assessed by PAGE under nondenaturing conditions. The Coomassie
blue-stained gel includes: lane 1, molecular mass markers (bands from
top to bottom: thyroglobulin, 669 kD; ferritin, 440 kD; catalase
232 kD; lactate dehydrogenase, 140 kD; albumin, 67 kD); lane 2, peak I
from Mono-Q (1 µg); and lane 3, peak II from Mono-Q (1 µg).
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Peaks I and II of the epimerase behaved as identical homogeneous
species when subjected to IEF on gels under denaturing conditions (Fig.
5A). The experimentally determined pI was
6.3, based on the assumption that a linear pH gradient was established
by the ampholytes, as compared with 6.0, as calculated from the amino acid composition. Peak I also focused primarily as a single compact species under nondenaturing conditions with a pI of 5.0, whereas peak
II contained an additional compact species with a pI of 4.9 (Fig. 5B).
These values were virtually the same (±0.1 pI unit) whether based on
the assumption of a linear pH gradient or on the internal protein
markers. Diffuse regions of staining bands were observed throughout the
pH gradient at values exceeding pH 5.0, presumably indicative of
dissociation and denaturation of the holoenzyme during IEF. These
patterns visualized by nondenaturing IEF were not altered by
rechromatography of the epimerase on Mono-Q, i.e. the single compact
band of peak I and the compact doublet of peak II persisted.
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| Figure 5.
IEF of recombinant epimerase under denaturing (A)
and nondenaturing (B) conditions. Lanes 1 and 2 in both Coomassie
blue-stained gels represent peaks I and II, respectively, from Mono-Q
chromatography (Fig. 2). Sample loads were 0.6 µg in A and 1.0 µg
in B. pI markers in B include (from top to bottom) human carbonic
anhydrase B (pI 6.55), bovine carbonic anhydrase B (pI 5.85),
-lactoglobulin A (pI 5.2), and soybean trypsin inhibitor (pI
4.55).
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Stability of Ru5P Epimerase
Whether in crude extracts or highly purified, the recombinant
epimerase is inherently unstable even at 2°C (Fig.
6). The instability did not correlate
with epimerase concentration and was not affected by glycerol,
exogenously added protein (BSA), cofactors (NADH, NAD, or ATP),
divalent metal ions (Mg2+ or
Zn2+), or EDTA (data not shown); instability was
exacerbated by Ru5P. Ethanol and G3P stabilized the enzyme but were
unable to reverse the spontaneous loss of activity.
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| Figure 6.
Inherent instability of recombinant epimerase and
effects of additives. All incubations were at 2°C in a pH 8.0 buffer
of 50 mM Bicine. With the exception the one data set
depicting centrifuged extract (250 µg total protein
mL1) without any additives ( ), all others depict
purified enzyme (50 µg mL1): no additives ( ), 10 mM G3P introduced at 0 time or after 2 h ( ), 20%
(v/v) ethanol ( ), 1 mM Ru5P ( ), and 10 mM
2-mercaptoethanol ( ). See text for additional details.
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In contrast to most intracellular enzymes, the epimerase was
drastically destabilized by 2-mercaptoethanol (Fig. 6). DTT also destabilized the enzyme, but the rate of activity loss was less pronounced; GSH was without effect (data not shown). Neither G3P nor
ethanol afforded protection against inactivation of the epimerase by 2-mercaptoethanol. Although sensitivity to thiols suggested reduction of a protein disulfide, neither GSSH nor
dehydroascorbate reversed the inactivation by 2-mercaptoethanol (data
not shown). Furthermore, spontaneous or 2-mercaptoethanol-induced
inactivation of the epimerase did not alter its electrophoretic
mobility or its IEF under nondenaturing conditions (data not shown).
Kinetic and Thermodynamic Parameters
Epimerase activity displayed a typical hyperbolic response to
increasing concentrations of Ru5P, from which a
Km of 0.22 mM and a
Vmax of 17,000 units
mg1 were calculated. The corresponding
kcat based on a subunit molecular mass of
25,000 D was 7083 s1, thereby establishing
kcat/Km as
3.2 × 107
M1 s1. G3P
(examined at both 1 and 5 mM) behaved as a competitive
inhibitor with a Ki of 0.9 mM.
At pH 8.0 and 25°C, the equilibrium ratio of Xu5P to Ru5P was 2.2, irrespective of epimerase concentration and preparation of epimerase
used. Under very similar conditions, an equilibrium constant of 1.5 has
been reported by use of impure epimerase from Lactobacillus
pentosus (Hurwitz and Horecker, 1956).
Authenticity of Spinach Recombinant Ru5P Epimerase
Our construct of a cDNA clone encoding the mature form of Ru5P
epimerase includes an initiation codon for Met in place of a codon for
Lys at the cleavage site of the transit peptide. This forecasts an
N-terminal sequence of MATSRVD for the recombinant epimerase, compared
with TSRVD for the epimerase isolated from spinach chloroplasts (Teige
et al., 1995, 1998). The N-terminal sequence of our purified
recombinant enzyme (a mixture of peaks I and II from Mono-Q) as
determined by Edman degradation was ATSRVD. Thus, the encoded
N-terminal Met was removed in E. coli, so the recombinant
enzyme differed from the authentic leaf protein merely by the presence
of a single Ala residue as an appendage at the N terminus.
Western blots of denaturing and nondenaturing PAGE gels showed that the
sizes of the recombinant epimerase subunit and the recombinant
epimerase holoenzyme closely match those of the authentic enzyme
present in extracts of fresh spinach leaves (Fig.
7, A and B). The antibody raised against
recombinant spinach Ru5P epimerase (a mixture of peaks I and II) did
not cross-react with the E. coli epimerase or with any other
protein in extracts of nontransformed E. coli (Fig. 7B, lane
3), but did cross-react with the component unique to peak II from
Mono-Q that was observed by IEF under nondenaturing conditions (Figs.
5B and 7C). Thus, this second component is a form of the recombinant
epimerase and does not reflect contamination with constitutive E. coli epimerase. The diffuse staining regions that were detected
with Coomassie blue (Fig. 5B) also cross-reacted strongly with the
antibody and displayed a rather prominent band with a pI of 5.7 (Fig.
7C). Because this value approached that of the fully denatured subunit
(pI 6.3), our previous supposition that the complex focusing pattern
reflects partial dissociation and denaturation of the epimerase was
reinforced.
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| Figure 7.
Western-blot analyses of purified recombinant
epimerase, E. coli extracts, and spinach leaf extract
subjected to SDS-PAGE (A), nondenaturing PAGE (B), or nondenaturing IEF
(C). In A, samples are as follows: lane 1, peak I from Mono-Q (0.12 µg); lane 2, centrifuged extract from transformed E. coli (2.5 µg); lane 3, centrifuged extract from spinach (20 µg); and lane 4, prestained molecular mass markers (bands from top to
bottom: myosin, 250 kD; phosphorylase b, 148 kD;
glutamic dehydrogenase, 60 kD; carbonic anhydrase, 42 kD; myoglobin, 22 kD; lysozyme, 17 kD; aprotinin, 6 kD). In B, samples are as follows:
lane 1, peak I from Mono-Q (15 ng); lane 2, peak II from Mono-Q (15 ng); lane 3, centrifuged extract from nontransformed E. coli (0.5 µg) (note the absence of cross-reactive material);
lane 4, centrifuged extract from transformed E. coli
(0.5 µg); and lane 5, centrifuged extract from spinach (15 µg). In
C, samples are as follows: lane 1, peak I from Mono-Q (0.5 µg); and
lane 2, peak II from Mono-Q (0.5 µg).
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DISCUSSION |
We elected to clone spinach rpe because previous clones
(Kusian et al., 1992; Nowitzki et al., 1995; Teige et al., 1995) have not led to efficient overexpression in a foreign host. In the only case
in which heterologous expression has been reported (Nowitzki et al.,
1995), transformation of E. coli with a plasmid harboring spinach rpe resulted in only a 3-fold elevation of epimerase
activity relative to activity indigenous to the host. This low level of activity may have reflected inherent instability of the recombinant enzyme rather than poor expression. By contrast, we observed a >350-fold elevation of activity with our expression system (0.7 unit
mg1 protein in extracts of nontransformed cells
versus 250 units mg1 protein in extracts of
transformed cells after induction), in which recombinant Ru5P epimerase
represents approximately 2% of the total soluble protein.
The extreme instability of the epimerase posed a hurdle to attaining a
high level of purity while preserving the catalytic activity. Our
discovery that G3P affords effective, long-term protection against
spontaneous loss of activity enabled us to clear this hurdle. By a
combination of maintaining the preparation at 2°C, including 10 mM G3P in all buffers used during the purification, and
minimizing the time for the chromatographic steps, extensive loss of
activity was averted. Some loss did occur during dialysis before Mono-Q
chromatography (with the preparation summarized in Table I, this loss
amounted to 11%, which is the worst case encountered among numerous
preparations from independent cultures during the past 1.5 years).
Final specific activities have ranged from 10,000 to 15,000 units
mg1. By comparison, the yeast and beef liver
epimerases, which are not inherently unstable, have specific activities
of approximately 7000 units mg1. Apart from the
dialysis step, the other variable with respect to final specific
activity may be the intracellular status of the enzyme at the time of
harvesting of the cultures. From sample application to pooling of
fractions, the DE52, hydroxyapatite, and Mono-Q chromatographic steps
were completed in 5, 1, and 2 h, respectively. The original
high-speed supernatant and the concentrated pools from each of the
three columns could be frozen in the absence of cryoprotectant and
stored indefinitely.
The specific activity of Ru5P epimerase recently purified from spinach
chloroplasts was observed to be only 180 units
mg1 (Teige et al., 1998). This extremely low
value, <2% of that attainable under our protective conditions,
probably reflected almost total inactivation of the enzyme wrought by 5 mM DTT, which was included in the initial extraction and
column buffers.
Spontaneous and thiol-induced inactivation of Ru5P epimerase reflect
distinct phenomena, because ethanol and G3P retard the former but not
the latter. Despite a rather extensive effort, we have not identified
any additives or conditions that even partially reverse spontaneous or
thiol-induced inactivation of the epimerase. Enhanced stability of the
enzyme in the presence of ethanol may be indicative of solvent-exposed
hydrophobic patches, which in vivo serve as sites for association with
other proteins or thylakoid membranes in situ. Although Ru5P epimerase
has not been found in various multienzyme complexes of Calvin cycle
enzymes (Sainis and Harris, 1986; Nicholson et al., 1987; Persson and
Johansson, 1988; Rault et al., 1993; Süss et al., 1993; Anderson
et al., 1995; Romanova and Pavolets, 1997), recent studies (Teige et
al., 1998) using immunogold electron microscopy have localized the epimerase to thylakoid membranes. As judged by kinetic analyses, the
binding of G3P and Ru5P by the epimerase is competitive. Therefore, the
opposite effects of these ligands on enzyme stability is likely explained by their inducing different conformational changes. Although
the thiol sensitivity of the enzyme immediately raises the prospect of
a structurally or catalytically important disulfide, experimental data
argue to the contrary. Thiol-induced inactivation is not reversed by
customary oxidants, and the inactivation does not result from
dissociation of subunits or any structural changes that can be detected
by a variety of electrophoretic analyses. Furthermore, none of the
three cysteinyl residues in each subunit of Ru5P epimerase are species
invariant, as would be expected if any were critical to structure or
catalysis.
Thiol sensitivity of the epimerase could be construed as reflective of
in vivo redox regulation via the Fd-thioredoxin system, which has been
well documented for several chloroplast enzymes (for reviews, see
Buchanan, 1991; Jacquot et al., 1997). However, our inability to
reverse the inactivation of Ru5P epimerase by 2-mercaptoethanol
precludes further speculation on this issue.
Most species of Ru5P epimerase are homodimeric; the only previous
exception is an octameric form of the enzyme isolated from spinach
chloroplasts (Teige et al., 1998). However, the authors of that study
cautioned that this may have been an artifact of purification because
the epimerase activity released from freshly ruptured chloroplasts
appeared to chromatograph as a dimer during gel filtration. Although we
cannot reconcile these disparate observations, our finding that highly
active recombinant spinach Ru5P epimerase is also octameric argues
against an anomalous quaternary structure arising during
purification. Furthermore, western-blot analysis of crude extracts of
spinach leaves clearly shows that the authentic epimerase co-migrates
with the recombinant form during nondenaturing PAGE (Fig. 7B),
indicative of in vivo assembly of the 25,000-D subunit into an
octameric holoenzyme. Whether octamers will prove typical of the
epimerase, as found throughout the plant kingdom, must await future
comparisons.
Despite the striking differences in subunit structure of spinach Ru5P
epimerase and the corresponding yeast and mammalian enzymes, their
kinetic parameters are rather similar. The
Km (Ru5P) of 0.22 mM for the
recombinant spinach enzyme compares with 0.2 to 1.5 mM for
other species reported (Hurwitz and Horecker, 1956; Wood, 1979;
Bär et al., 1996), and the Vmax of
the former is about twice as great as the highest
Vmax reported among the latter (Terada et
al., 1985; Bär et al., 1996). The catalytically impaired preparation isolated from spinach chloroplasts exhibited a similar Km of 0.25 mM (Teige et al.,
1998). Thus, the DTT-induced inactivation of the epimerase does not
alter its apparent affinity for substrate.
We have not been able to determine the molecular basis of
microheterogeneity displayed by the recombinant epimerase when
chromatographed on Mono-Q (Fig. 2) or isoelectric focused under
nondenaturing conditions (Figs. 5B and 7C). Obvious possibilities such
as contamination by E. coli Ru5P epimerase or incomplete
removal of the N-terminal Met residue encoded by the gene construct are
excluded. With respect to the former, the antibody raised against the
recombinant preparation does not cross-react with E. coli
epimerase, and furthermore, >100 mM NaCl is required to
elute the E. coli enzyme from the DEAE column compared with
<50 mM NaCl for the recombinant enzyme. The only residue
detected in the first cycle of Edman degradation is Ala, without even a
trace of Met, thereby ruling out mixed N-terminal processing as a basis
for microheterogeneity. We also dismiss partial dissociation of
subunits as an explanation because the preparations always migrated as
a single species during nondenaturing PAGE. If the microheterogeneity
is caused by size or charge differences at the subunit level, they are
below the threshold for detection by SDS-PAGE and IEF under denaturing
conditions. The subtlety of the basis of microheterogeneity is
emphasized by the very small difference of only 0.1 pI unit between the
two bands (5.0 versus 4.9) observed by IEF of the native enzyme. In
contrast, a difference of only one formal charge per subunit is
calculated to alter its pI by 0.35 unit. We speculate that we may be
encountering three discrete conformations of the enzyme, a pair that
co-chromatograph on Mono-Q (the two components in peak II that focus
with pI values of 5.0 and 4.9, respectively), and a different pair that
co-focus on native IEF (the single component in peak I, which exhibits a pI of 5.0, and the component in peak II with a pI of 5.0). Future studies of reversible denaturation may provide an avenue for clarifying this situation.
In summary, to our knowledge we have designed the first high-level
expression system for an rpe clone from any source.
Subsequent development of a facile purification scheme for the
recombinant spinach Ru5P epimerase enabled us to characterize
structural, catalytic, and stability properties of the first highly
active Ru5P epimerase of photosynthetic origin obtained.
| |
FOOTNOTES |
1
This research was supported by the Office of
Biological and Environmental Research, U.S. Department of Energy, under
contract no. DE-AC0596OR22464 with the Lockheed Martin Energy Research Corporation.
*
Corresponding author; e-mail ffh{at}ornl.gov; fax 1-423-574-0793.
Received April 30, 1998;
accepted June 12, 1998.
2
cDNA for spinach rpe (Nowitzki et
al., 1995) had not been reported at the time we initiated our
studies.
3
Residue numbers refer to the mature recombinant
enzyme, the N-terminal Ala of which corresponds to position 49 of the
transit protein.
| |
ABBREVIATIONS |
Abbreviations:
G3P, DL--glycerophosphate.
R5P, D-Rib-5-phosphate.
Ru5P, D-ribulose-5-phosphate.
Ru5P epimerase, D-ribulose-5-phosphate 3-epimerase.
Xu5P, D-xylulose-5-phosphate.
| |
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Abstract
Introduction