From the Section of Microbiology, Division of Biological Sciences, University of California, Davis, California 95616
Received for publication, November 20, 2000, and in revised form, December 11, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glucose-6-phosphate dehydrogenase (G6PD), encoded
by zwf, is essential for nitrogen fixation and dark
heterotrophic growth of the cyanobacterium Nostoc
punctiforme ATCC 29133. In N. punctiforme, zwf is part of a four-gene operon transcribed in the order
fbp-tal-zwf-opcA. Genetic analyses indicated that
opcA is required for G6PD activity. To define the role of
opcA, the synthesis, aggregation state, and activity of
G6PD in N. punctiforme strains expressing different amounts
of G6PD and/or OpcA were examined. A single tetrameric form of G6PD was
consistently observed for all strains, as well as for recombinant
N. punctiforme His-G6PD purified from Escherichia coli, regardless of the quantity of OpcA present. However,
His-G6PD and the G6PD of strain UCD 351, which lacks OpcA, had low
affinities for glucose 6-phosphate (G6P) substrate
(Km(app) = 65 and 85 mM,
respectively) relative to wild-type N. punctiforme G6PD
(Km(app) = 0.5 mM). Near wild-type
affinities for G6P were observed for these enzymes when saturating
amounts of His-OpcA- or OpcA-containing extract were added. Kinetic
studies were consistent with OpcA acting as an allosteric activator of G6PD. A role in redox modulation of G6PD activity was also indicated, because thioredoxin-mediated inactivation and reactivation of His-G6PD
occurred only when His-OpcA was present.
Cyanobacteria are members of a phylogenetically cohesive group of
prokaryotes that perform oxygenic photosynthesis and assimilate CO2 via the reductive pentose phosphate (Calvin) cycle.
Excess photosynthate produced in the light is stored as
glycogen, which is catabolized to provide maintenance energy during
periods of darkness. Although most cyanobacteria are restricted to this
photoautotrophic mode of growth, some, such as the filamentous
heterocyst-forming isolate Nostoc punctiforme ATCC 29133, exhibit greater nutritional versatility. When provided with exogenous
sugars such as fructose or glucose, N. punctiforme is
capable of both photoheterotrophic growth in the light and
heterotrophic growth in the dark.
The oxidative pentose phosphate
(OPP)1 cycle is assumed to be
the major route for carbon catabolism in cyanobacteria, both in
vegetative cells for the breakdown of glycogen or exogenously supplied
sugars, and in nitrogen-fixing heterocysts, where it provides a
reductant (NADPH) required by nitrogenase (1-3). Regulation of the
activity of glucose-6-phosphate dehydrogenase (G6PD; E.C. 1.1.1.49),
the enzyme controlling the entry of carbon into the cycle, is complex
and poorly understood. Metabolites such as NADPH, ATP, glucose
6-phosphate (G6P), glutamine, and ribulose bisphosphate have been
implicated in its regulation (4, 5) as has thioredoxin, which may
reductively inactivate and oxidatively reactivate the enzyme in the
light and dark, respectively, to prevent futile cycling, which would
occur if the Calvin and OPP cycles operated simultaneously (5-8). G6PD
has been purified from the filamentous, heterocyst-forming
cyanobacteria Anabaena variabilis (5) and Anabaena sp. strain PCC 7120 (Anabaena 7120) (4,
8). Although the A. variabilis enzyme is a 250-kDa tetramer
(5), the Anabaena 7120 enzyme is found in multiple
aggregation states. Three forms (M1, M2, and
M3; 120, 240, and 345 kDa, respectively) with different catalytic activities were initially reported for Anabaena
7120 G6PD (4); the 240-kDa M2 form was by far the most
prevalent, but the larger M3 form appeared to be more
active. More recently, the Anabaena 7120 enzyme was reported
to exist in two aggregation states (250 and 750 kDa) (8). The
equilibrium between the different forms of the Anabaena 7120 enzyme depends upon pH, the concentration of the enzyme in solution,
and the presence or absence of substrates or other effectors. Multiple
reactive bands have also been observed in extracts from
Anabaena 7120 and some other cyanobacterial strains after
native polyacrylamide gel electrophoresis (PAGE) and staining of the
gels for G6PD activity (9). The sizes, numbers, and intensities of the
bands vary with the strain and with growth conditions.
A genetic analysis of the physiological role of G6PD and the OPP cycle
in N. punctiforme was recently performed (10). In N. punctiforme, zwf, the gene encoding G6PD, is part of a
four-gene operon
(fbp-tal-zwf-opcA)2
with multiple transcriptional start points and complex transcriptional regulation (11). Insertional inactivation of zwf resulted in essentially complete loss of G6PD activity and rendered the
zwf mutant incapable of nitrogen fixation or dark
heterotrophic growth, indicating that G6PD is required for both of
these processes (10). Inactivation of opcA gave rise to a
mutant with the same phenotype. The opcA mutant was fully
complemented by opcA supplied in trans on a multicopy
plasmid, whereas complementation of the zwf mutant with
zwf on the same multicopy plasmid abolished the growth
defects but restored G6PD activity to only 25% of wild-type levels. If both zwf and opcA were present on the multicopy
plasmid, however, the G6PD activity of the complemented zwf
mutant was increased 20-fold over wild-type levels. These results imply
that the downstream gene opcA is required for the synthesis
or activation of fully functional G6PD.
The opcA gene has since been identified in all other
cyanobacteria examined. Outside of the division Cyanobacteria, a
derived amino acid sequence with significant similarity across the
entire length of the polypeptide has not been observed. A limited
similarity to restricted regions of the polypeptide can be detected in
derived sequences from other microorganisms by BLAST analysis
(available from the National Center for Biotechnology Information on
the Web) (12). In Synechococcus sp. PCC 7942 (Synechococcus 7942) (13) and Anabaena 7120 (available from Kazusa DNA Research Institute on the Web),
opcA is located directly downstream from zwf,
whereas the two genes are unlinked in Synechocystis sp.
strain PCC 6803 (also available from the National Center for
Biotechnology Information on the Web). Insertional inactivation of
opcA in Synechococcus 7942 resulted in a mutant
that had lost G6PD activity and was no longer viable in the dark (9), a
phenotype consistent with that observed for N. punctiforme
(10). On the basis of G6PD activity staining of mutant and wild-type
cell extracts on native PAGE gels, it has been proposed that OpcA
catalyzes the formation of active G6PD oligomers in
Synechococcus 7942 (9). We report here the results of
further experiments undertaken with N. punctiforme that are
consistent with OpcA acting as an allosteric activator of G6PD and not
as a chaperone for the assembly of G6PD oligomers.
Strains, Culture Conditions, and Cell Lysis--
Plasmids and
bacterial strains used in this study are shown in Table
I. N. punctiforme 29133 and
Anabaena 7120 were cultured in modified Allen and Arnon
medium (14) at 25 °C in an illuminated (9 watts/m2) New
Brunswick incubator with shaking at 150 rpm. The medium was
supplemented with 2.5 mM NH4Cl and 5 mM MOPS, pH 7.8, with 50 mM fructose or
glucose, or with ampicillin (Ap) or neomycin (Nm) at 10 µg/ml when
appropriate. When darkness was required, culture flasks were wrapped in
multiple layers of aluminum foil to exclude light. Cells were typically
harvested after 7-10 days when culture densities reached 2-3
µg of chlorophyll a (Chl a)/ml.
Extracts used for SDS and native PAGE and enzyme assays were prepared
from cells grown as described above and harvested by centrifugation
at 6000 × g, 4 °C. Cell pellets were washed once in
50 mM Tris maleate buffer (pH 6.5) containing 10 mM MgCl2, resuspended in the same buffer, and
broken with several passages through a chilled French pressure cell at
15,000 p.s.i. Extracts were clarified by centrifugation for 5 min at
16,000 × g, 4 °C prior to use. Protein assays and
determinations of the Chl a content of cyanobacterial
strains were performed as described previously (15).
Strain Construction and Protein Purification--
Routine DNA
manipulations in Escherichia coli strain DH5
Affinity-tagged G6PD and OpcA proteins were prepared using the pET
system of Novagen or the pMAL protein fusion and purification system of
New England BioLabs. DNA fragments containing the N. punctiforme
zwf or opcA genes were obtained using the polymerase chain reaction (PCR) with pSCR115 as a template and the following primers: 5'-ATGGTTAGTCTGCTAGAAAATCCCTTGC-3' (His-G6PD forward) and 5'-GCTCTAGAGTCTGCGCCAGCGACGGCCATCC-3' (His-G6PD reverse), or
5'-ATGACAAAATCCAAAATCTCAAATCCAAAACTTCC-3' (MBP-OpcA and His-OpcA forward) and 5'-GCTCTAGACGGTGTATACACAAGTCGTTAAAACCCG-3' (MBP-OpcA reverse) or 5'-CGCGCTCGAGGTTAGGTCTAAGTTAATTCG-3' (His-OpcA reverse). PCR amplifications were performed in a PerkinElmer Cetus thermal cycler with AmpliTaq polymerase. The 1.5-kb PCR product containing zwf was initially cloned into the EcoRV site of
pBluescript II KS(+). Digestion with BamHI and
HindIII released a 1.5-kb fragment containing
zwf, which was inserted into the BamHI and
HindIII sites of the expression vector pET-28a(+) to
generate pSCR197. pSCR195, used to generate MBP-OpcA for antibody
production, was constructed by cloning a 1.6-kb PCR fragment containing
opcA into the EcoRV site of pBluescript II KS(+).
This fragment was removed from pBluescript II KS(+) by digestion with
SalI and PstI and was then ligated into the
SalI and PstI sites of pMAL-c2. A 1.4-kb PCR
fragment containing opcA was digested with XhoI
and inserted into the Ecl136II and XhoI sites of
pET-28a(+) to generate pSCR352, which was used to produce His-OpcA.
MBP-OpcA was soluble when expressed in E. coli DH5 PAGE and Immunoblots--
SDS and native PAGE and Coomassie Blue
staining were performed according to standard protocols (20) using
either the Bio-Rad Mini-Protean II system or the larger Bio-Rad Protean
system. Rabbit polyclonal antisera were raised against purified
His-G6PD or MBP-OpcA by standard techniques (21) and were used at a
dilution of 1:400. Immunoblotting was performed as described previously
(22). Bound primary antibody was detected either with horseradish
peroxidase-conjugated goat
The molecular masses of His-G6PD and G6PDs in cyanobacterial extracts
were determined by Ferguson analysis using 5, 6, and 7% acrylamide
native PAGE gels. Standards used for native PAGE were obtained from
Sigma Chemical Co. and included the following: bovine serum albumin (66 and 132 kDa), G6PD Activity Assay--
G6PD activity was assayed by a
modification of the method of Schaeffer and Stanier (4), in which the
standard assay NADP concentration was increased from 0.5 to 2.0 mM. The standard assay mixture also contained 50 mM Tris maleate, pH 7.5, 10 mM
MgCl2, and 5 mM G6P (dipotassium salt). Where
indicated, the G6P and NADP concentrations were varied for the
determination of kinetic parameters. The reaction was initiated by the
addition of cell extract or purified protein, and the reduction of NADP
at room temperature was followed by monitoring the increase in
absorbance at 340 nm, typically for 1 min. To ensure that initial
velocities were measured, all assays were determined to be in the
linear range with respect to protein concentration, time, and substrate utilization. With crude cell extracts, in which the enzyme
concentration varied, at least three total protein concentrations were
surveyed for linearity before extended assays were done at the selected protein concentration. For purified His-G6PD, activity in standard assays was linear between at least 0.2 and 2.0 µg of protein and 0.5 µg was typically used. The quantity of G6P and NADP substrate consumed was calculated at all substrate, protein, and effector concentrations used. For determinations of Km(app)
for NADP, less than 5% of the initial concentrations of NADP or G6P were consumed and for determinations of Km(app) for
G6P, the amounts of G6P and NADP consumed were always less than 2% of
the initial concentrations. In all experiments, at all concentrations of protein, substrates, and effector, changes in
A340 were linear with time, implying that any
potential end product inhibition by NADPH or 6-phosphogluconolactone
was inconsequential. Kinetic data were analyzed using KaleidaGraph v.
3.08. Spirulina thioredoxin and the chemical oxidants and
reductants used in redox modulation experiments were obtained from
Sigma Chemical Co.
Growth Condition-dependent Activity and Aggregation
State of G6PD--
To determine whether growth conditions affected the
activity or aggregation state of N. punctiforme G6PD, cells
were cultured with ammonium in the light (photoautotrophic conditions),
without ammonium in the light (photoautotrophic, nitrogen-fixing
conditions), or in the dark in the absence of ammonium and presence of
50 mM fructose (heterotrophic, nitrogen-fixing conditions).
Cell extracts prepared from each culture were first assayed for G6PD
activity and were then separated by electrophoresis on 10% acrylamide
native PAGE gels, blotted to nitrocellulose membrane, and incubated
with Molecular Mass and Monomer Composition of Native
G6PD--
N. punctiforme G6PD consistently migrated as a
single band with a molecular mass that appeared to be between 200 and
545 kDa based on the positions of native PAGE standards, whereas OpcA migrated as a single band near the 66-kDa marker (Fig. 1). Because the
predicted molecular masses of the N. punctiforme zwf and
opcA gene products are 58 and 51 kDa, respectively, these
results implied that the G6PD band was an oligomer and the OpcA band
was a monomer. G6PD and OpcA from N. punctiforme extracts
were never observed to comigrate on native PAGE gels (e.g.
compare lanes 1-3 with lanes 4-6 in Fig. 1). To
confirm the implication that OpcA was not associated with the G6PD
oligomer in N. punctiforme cell extracts, immunoprecipitation experiments were performed using either
Ferguson analysis was used to more precisely determine the molecular
mass of the G6PD oligomer. Samples containing N. punctiforme cell extract, Anabaena 7120 cell extract (control), or
N. punctiforme His-G6PD purified from E. coli
were loaded on a series of native PAGE gels (5, 6, and 7% acrylamide)
along with native protein standards. After electrophoresis, the
proteins were visualized with Coomassie Blue (standards) or G6PD
activity stain (samples containing G6PD). A single strong band that
appeared within 15 min of G6PD activity staining was observed for
His-G6PD and in N. punctiforme and Anabaena 7120 extracts (Fig. 2). A faint lower molecular mass band was occasionally detected in Anabaena
7120 extract after several hours of staining; no additional bands were observed with His-G6PD or N. punctiforme extracts after
prolonged staining, nor were additional bands detected with extracts
from cells grown with glucose or fructose (data not shown).
RF values were calculated for each protein, and the
molecular masses of the cyanobacterial G6PDs were determined as shown
in Fig. 3 (A and
B). The molecular masses of the G6PDs in N. punctiforme and Anabaena 7120 cell extracts were 200 and 263 kDa, respectively, whereas the molecular mass of His-G6PD was
229 kDa. There was no change in the migration of His-G6PD upon the
addition of His-OpcA, nor was the aggregation state of His-G6PD altered
by increasing its concentration in solution to 2.5 mg/ml, by changing
the buffer pH from 7.9 to 6.5 or by incubating it with G6P or NADP
(data not shown).
Effect of the Stoichiometry Between OpcA and G6PD on G6PD Activity
and Aggregation State--
To determine whether differences in the
amount of OpcA present in the cell affected the quantity, aggregation
state, or activity of N. punctiforme G6PD, G6PD assays and
quantitative Western blots were performed with extracts from several
strains that produced amounts of OpcA and G6PD that differed from those
normally found in wild-type N. punctiforme. The results are
summarized in Table II. Wild-type
N. punctiforme extract typically contained 1 or 2 mg of OpcA
and G6PD monomers per g of cell protein when the cells were cultured
with ammonium or dinitrogen, respectively. The aggregation state of
G6PD did not change when the stoichiometry between OpcA and G6PD was
altered (Table II); in all strains that carried one or more intact
copies of the zwf gene, G6PD monomers were synthesized and
assembled into a single aggregation state that corresponded to the
200-kDa form found in wild-type N. punctiforme. The G6PD
activity varied, however. In strain UCD 351, which produced 15 times
more G6PD than the wild-type, but lacked OpcA, G6PD activity was nearly
9-fold lower than in wild-type. Activity was at baseline levels in
strains UCD 466 and UCD 341, which lacked G6PD but differed in their
OpcA content. In strains that produced both G6PD and OpcA, G6PD
activity depended on the total quantity of G6PD present and on the
stoichiometry between OpcA and G6PD: In strain UCD 482, which
synthesized normal amounts of G6PD in the presence of elevated amounts
of OpcA, G6PD activity was similar to that of wild-type. Near-wild-type
G6PD activity was also observed for strain UCD 364, which overproduced
G6PD in the presence of wild-type amounts of OpcA. A large (23-fold)
increase in G6PD activity was observed only for strain UCD 348, which
overproduced both OpcA and G6PD.
The experiments described above implied that G6PD synthesized in the
absence of OpcA was assembled into its native aggregation state but was
still relatively inactive. To determine whether the activity of this
inactive G6PD could be restored by the addition of OpcA, extract from
strain UCD 351 (G6PD overproduced, no OpcA) was mixed with extract from
strain UCD 466 (no G6PD, OpcA overproduced). In the experiment shown in
Fig. 4, the amount of strain UCD 351 extract was held constant, and increasing amounts of strain UCD 466 extract were added until no further change in G6PD activity was
observed. The G6PD activity of the strain UCD 351 extract increased
nearly 20-fold, from 0.007 µmol·min
Kinetic Constants of G6PD Measured in the Absence and Presence of
OpcA--
Additional experiments were performed with cell extracts and
the purified proteins to determine the nature of the defect in N. punctiforme G6PD synthesized in the absence of OpcA. A comparison of kinetic parameters revealed that the affinity of G6PD for G6P substrate was ~30-fold lower when OpcA was absent. When the amount of
cell extract was held constant and the concentration of G6P was varied,
a sigmoidal rate versus substrate concentration curve was
obtained with strain UCD 351 extract, whereas a hyperbolic curve was
obtained with wild-type N. punctiforme extract (Fig. 5A). A sigmoidal curve was
also obtained with His-G6PD (Fig. 5B). The apparent
Km for G6P (S0.5) was
determined to be 0.5 ± 0.1 mM for wild-type N. punctiforme G6PD, 85 mM for strain UCD 351 G6PD, and
65 ± 10 mM for His-G6PD (Table
IV). Near-wild-type affinities for
substrate were restored to His-G6PD and strain UCD 351 G6PD when
saturating amounts of His-OpcA were added to the assay (Fig.
5B and Table IV). As illustrated in Fig. 5B, the shift in apparent Km was unaccompanied by a change
in Vmax. The kinetics of NADP cofactor binding
were also examined (Table IV). At saturating G6P concentrations, NADP
binding curves were nearly hyperbolic. In the absence of OpcA, the
apparent Km for NADP was 48 µM for
strain UCD 351 extract G6PD and 49 ± 4.2 µM for
His-G6PD. Upon the addition of His-OpcA, a 2-fold reduction in the
apparent Km for NADP occurred and
Km(app) values nearly identical to those of
wild-type N. punctiforme extract were obtained.
Effect of Redox Modulators on OpcA and G6PD Activity--
The
activity of cyanobacterial G6PD is reported to depend upon its
oxidation-reduction state; therefore, the potential for redox
regulation of His-G6PD was examined. First, chemical oxidizing or
reducing agents were added to His-G6PD samples to a final concentration of 5 or 50 mM. The samples were incubated 30 min on ice and
assayed for G6PD activity. Neither the oxidizing agents sodium
tetrathionate (STT), diamide, hydrogen peroxide, or oxidized
glutathione nor the reducing agents dithiothreitol (DTT) or reduced
glutathione affected the activity of His-G6PD that had not been
previously activated by His-OpcA; however, when DTT was added at high
concentration (50 mM) to a mixture containing both His-G6PD
and His-OpcA, the G6PD activity of the mixture declined 77% after 5 min and 96% after 30 min. No activation of His-G6PD occurred when
His-OpcA was incubated with 50 mM DTT for 30 min prior to
the mixing of the two proteins.
To determine whether similar results could be obtained with
thioredoxin, Spirulina thioredoxin that had been reduced or
oxidized with 2 mM DTT or 2 mM STT,
respectively, was added to His-G6PD or His-OpcA. Like the
chemical-oxidizing and -reducing agents, reduced or oxidized
thioredoxin had no effect on the activity of His-G6PD when it was added
in the absence of His-OpcA (data not shown). However, when reduced
thioredoxin was added to His-OpcA, His-G6PD added subsequently to the
His-OpcA plus reduced thioredoxin mixture was no longer activated (Fig.
6). The G6PD-activating ability of
His-OpcA was restored when STT was added to the His-OpcA/thioredoxin mixture at a concentration that was twice the DTT concentration (Fig.
6), and this reduction/oxidation cycle of inactivation/activation could
be repeated in the same sample. When thioredoxin was omitted from the
His-OpcA/thioredoxin mixture, the low concentrations of DTT and STT
appeared to have little effect, and activation of His-G6PD by His-OpcA
was always observed (Fig. 6).
The opcA gene was previously shown to be required for
G6PD activity in N. punctiforme (10) and
Synechococcus 7942 (9), and it has been proposed that OpcA
is involved in the assembly of active G6PD oligomers (9). Using
N. punctiforme strains that lack OpcA or express amounts of
G6PD and OpcA that differ from wild-type, we have shown that the
assembly of G6PD into its native tetrameric form occurs in N. punctiforme even in the absence of OpcA or when the stoichiometry
between OpcA and G6PD is greatly altered. Although N. punctiforme G6PD is assembled correctly in the absence of OpcA, it
is essentially inactive, because it has a very low affinity for G6P
substrate. Our kinetic studies with G6PD and OpcA indicate that OpcA
may function as a positive allosteric effector that shifts the
Km for G6P substrate to a physiologically relevant
value. OpcA may also be involved in redox regulation of G6PD activity,
because redox modulators affected G6PD activity only when OpcA was present.
During nitrogen fixation and dark heterotrophic growth, the cellular
demand for reductant and, ultimately, energy from the OPP pathway is
high, and increased transcription of the zwf and opcA genes of N. punctiforme has been documented
(11). In extracts from N. punctiforme grown under these
conditions, G6PD activity was elevated above the levels detected in
cells grown photoautotrophically with ammonium. Although there was
increased synthesis of G6PD and OpcA, no change in the aggregation
state of G6PD was detected (Fig. 1). Thus, rather than producing more
highly aggregated and catalytically more active forms of G6PD when
carbon flow through the OPP pathway increased, N. punctiforme simply synthesized more OpcA and additional G6PD
oligomers of a single aggregation state (Fig. 1).
G6PD purified from prokaryotic and eukaryotic organisms most often
exists as a homodimer or homotetramer, although larger oligomers have
been described (18). Enzyme concentration, pH, ionic strength, and the
presence of divalent cations, G6P, or NADP have all been reported to
affect the activity and aggregation state of the enzyme in
vitro (18). Several aggregation states that differ in their
kinetic properties have been described in Anabaena 7120 (4,
8), and multiple bands have been detected in extracts from several
cyanobacterial strains on native PAGE gels stained for G6PD activity
(9). Ferguson analysis in our laboratory revealed that the molecular
masses of the G6PD in N. punctiforme and Anabaena
7120 cell extracts were 200 and 263 kDa respectively, whereas N. punctiforme His-G6PD synthesized in and purified from E. coli was 229 kDa (Fig. 3B). The value of 263 kDa we
obtained for the enzyme in Anabaena 7120 extract is in good
agreement with the 240-kDa value obtained by Schaeffer and Stanier (4)
for the predominant M2 form of the enzyme, as well as with
the 250-kDa value obtained for the smaller of the two forms observed by
Gleason (8). There was no indication that OpcA comigrated with native
G6PD (Fig. 1), and the two proteins were not coimmunoprecipitated from
N. punctiforme extracts; therefore, it is unlikely that the
G6PD oligomer contains OpcA or that any stable protein complex
containing both OpcA and G6PD exists in N. punctiforme. From
sequence analysis, the G6PD monomers from both N. punctiforme and Anabaena 7120 are predicted to be ~58 kDa, whereas the His-G6PD monomer is 62 kDa. We conclude from these
data that the N. punctiforme, and most likely the
Anabaena 7120 and A. variabilis, native enzymes
are homotetramers with slightly different migratory properties. As
discussed below, we speculate that the higher aggregation states that
have been observed in vitro do not reflect the native state
of the protein in vivo.
Western blots showed that G6PD was assembled into its characteristic
tetrameric 200-kDa form in every N. punctiforme strain that
contained an intact zwf gene (Table II). N. punctiforme G6PD produced as a His-tagged protein in E. coli was found in the same aggregation state (Fig. 3, A
and B), although E. coli appears by BLAST
analysis to lack any protein with similarity to OpcA (12). There was
also no change in the aggregation state of purified His-G6PD after
activation by His-OpcA. Collectively, these results strongly imply that
OpcA is not required for assembly of the N. punctiforme G6PD
homotetramer. On native PAGE gels stained for G6PD activity, an
increase in the intensity of the highest molecular mass G6PD band was
observed upon mixing of extract from wild-type Synechococcus
7942 with extract from a Synechococcus 7942 strain lacking
OpcA (see Fig. 5 in Ref. 9). In light of the results presented here,
this increase in staining intensity is most likely to have been due to
the activation of previously assembled, catalytically inactive
tetrameric G6PD by OpcA rather than to OpcA-mediated assembly of
inactive G6PD monomers into catalytically active oligomers, as was
proposed (9).
OpcA was clearly required for the optimal catalytic activity of
N. punctiforme G6PD, and its effect was to increase the
affinity of G6PD for the G6P substrate ~30-fold to a physiologically
relevant value (Table IV). OpcA also affected NADP cofactor binding,
because a 2-fold increase in the affinity for NADP occurred for strain UCD 351 G6PD or His-G6PD in the presence of saturating His-OpcA. G6PD
activity assays of extracts from strains that overproduced G6PD (Table
II, strains UCD 351, UCD 364, and UCD 348) showed that, when G6PD was
present in excess, it was the amount of OpcA that determined G6PD
activity. As illustrated in Fig. 4 and Table III, the G6PD in strain
UCD 351 was activated by the addition of OpcA from strain UCD 466 extract. Both strain UCD 466 extract and purified His-OpcA were able to
activate strain UCD 351 G6PD and His-G6PD (Table III), indicating that
it was OpcA that was responsible for the activation of G6PD and not
some other component in N. punctiforme extract whose
activity depended on the presence of OpcA. For strain UCD 351 G6PD, the
OpcA concentration required for half-maximal activation of G6PD was
much lower than that required for half-maximal activation of His-G6PD
(Table III). The His-G6PD tetramer may have interacted less effectively
with OpcA than the native G6PD tetramer if the His tag made it less
accessible to OpcA.
The affinity of cyanobacterial G6PD for G6P in vitro is
reported to vary with the pH, with the concentration of the enzyme in
solution, and with its redox state and aggregation state (4, 5, 8, 23).
We were able to alter the affinity of purified His-G6PD for substrate
only by adding OpcA, but the shift in apparent Km
that occurred after OpcA was added (Fig. 5B and Table IV)
resembles the shift that others have observed when dilute purified
cyanobacterial G6PD was concentrated in solution (4, 8). Most recently,
for example, Gleason (8) reported that G6PD purified from
Anabaena 7120 cultured with nitrate was in a "high
activity" form when concentrated in solution ( To prevent simultaneous operation of the oxidative and reductive
pentose phosphate cycles, cyanobacterial and chloroplast G6PDs are
active in the dark when they are in an oxidized state and are inactive
in the light when they are in a reduced state, whereas the converse is
true of key enzymes of the reductive pentose phosphate cycle (6, 24).
The inactivation of G6PD is mediated by thioredoxin (5, 7, 8, 25) and
occurs via covalent redox modification of specific regulatory cysteine
residues (19). The location of the conserved cysteine residues differs
between the cyanobacterial and chloroplast enzymes (19, 26), and plants appear to lack any protein with similarity to cyanobacterial OpcA (BLAST search) (12), therefore, regulation of the two enzymes may be quite different. Because the oxidized and reduced forms of G6PD
have been shown to differ in their affinities for G6P (19, 23, 27), we
initially considered the possibility that strain UCD 351 G6PD and
His-G6PD were in a reduced and inactive form and that the shift in
apparent Km we observed was due to oxidative
reactivation of the enzyme upon the addition of OpcA. Two lines of
evidence now make this hypothesis unlikely: First, we were unable to
oxidatively activate His-G6PD either with oxidized thioredoxin or with
chemical oxidants such as STT, diamide, oxidized glutathione, and
hydrogen peroxide, which have previously been reported to activate G6PD
(7, 27); thus, oxidation by OpcA also seems unlikely. Second, the
experiments with When His-OpcA was incubated with reduced thioredoxin before it was
mixed with His-G6PD, no activation of His-G6PD occurred (Fig. 6);
activation of His-G6PD by His-OpcA occurred only after excess sodium
tetrathionate was added to the His-OpcA plus reduced thioredoxin
mixture. There are two possible explanations for these results: The
first is that the His-OpcA was unaffected by reduced thioredoxin, but
once His-G6PD had been activated by His-OpcA, it immediately became
sensitive to redox modulation by thioredoxin; the second is that
His-OpcA itself was reversibly inactivated by thioredoxin and could not
activate His-G6PD when it was in a reduced state. Because the
activation of His-G6PD by His-OpcA occurred very rapidly, we could not
distinguish between these two possibilities by catalytic assays. Like
other cyanobacterial G6PDs, N. punctiforme G6PD contains
cysteine residues in conserved positions that may be involved in redox
modulation of enzyme activity (26). The N. punctiforme OpcA
sequence also contains cysteine residues; of the nine present, those at
positions 183, 195, 396, 401, and 407 are conserved in five other
cyanobacterial sequences with homology to OpcA and therefore appear to
be the most likely targets for thioredoxin-mediated redox modulation of
OpcA activity, if it does occur. (The N. punctiforme,
Synechococcus 7942, and Synechocystis 6803 OpcA
sequences are available on the National Center for Biotechnology
Information web site; the Prochlorococcus marinus and marine
Synechococcus sp. strain WH8102 OpcA sequences are both
available at the U. S. Department of Energy Joint Genome Institute web
site; and the Anabaena 7120 OpcA sequence is available at
the Kazusa DNA Research Institute web site.) Detailed kinetic studies and site-directed mutagenesis of the cysteine residues in G6PD
and OpcA should allow us to determine whether reduced thioredoxin
inactivates one or both of these proteins.
The physiological role of OpcA and the exact nature of its interaction
with G6PD remain to be determined. N. punctiforme strains lacking OpcA have low levels of G6PD activity and exhibit defects in
nitrogen fixation and dark heterotrophic growth. The kinetic data
presented here are most consistent with a role for OpcA as an
allosteric activator of cyanobacterial G6PD that shifts the equilibrium
between the T- and R-states of the enzyme toward the higher affinity
R-state. We have also shown, however, that, for wild-type N. punctiforme cells, changes in growth conditions did not lead to
disproportionate changes in the amounts of OpcA and G6PD protein. If
OpcA is an allosteric effector of G6PD, it is unclear how it would
function effectively if its concentration in the cell does not change
significantly under conditions where regulation of enzyme activity is
required. One possibility is that OpcA itself is modified in some
manner, such that all or a portion of it is unable to interact with
G6PD under conditions when low enzyme activity is desirable. The redox
modulation experiments we have performed are consistent with the idea
that OpcA is reductively inactivated by thioredoxin, although other
forms of protein modification can also be considered.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Plasmids and bacterial strains
-MCR were
performed according to standard procedures (16). DNA-modifying enzymes
were purchased from New England BioLabs and Life Technologies, Inc. and
were used according to the manufacturers' instructions. Large scale
plasmid preparations were obtained using a commercial kit (Qiagen).
N. punctiforme strain UCD 466 (zwf::PpsbAnptII) was
constructed by digesting pSCR115 with PmlI to remove a
0.7-kb fragment from the zwf gene. This fragment was
replaced with a 1.1-kb-blunted XbaI fragment from pRL448
containing PpsbAnptII (encoding Nm resistance) to
generate pSCR346, which was digested with DraI and
XbaI. The 6.7-kb fragment containing the
nptII-interrupted zwf gene was ligated to the
sacB-containing conjugatable vector pRL271, which had been
digested with Ecl136II and XbaI. In the resulting
plasmid, pSCR347, PpsbAnptII is in the same orientation as zwf and transcription of opcA
occurs. Introduction of pSCR347 into wild-type N. punctiforme by conjugation, selection for Nm- and
sucrose-resistant exconjugants, and confirmation of the insertional
inactivation by Southern blotting was performed as described previously
(10, 14). Strain UCD 482 was constructed by introducing pSCR121 into
wild-type N. punctiforme via electroporation as described
(10). The construction of strains UCD 341, 348, 351, and 364 is
described elsewhere (10, 17).
-MCR,
whereas a portion of each of the His-tagged proteins was present in
inclusion bodies when expressed in E. coli BL21(DE3). In
each case, the soluble protein was purified by affinity chromatography
according to the kit manufacturers' instructions, with
phenylmethylsulfonyl fluoride (1 mM) added to all buffers
used during purification. Purified proteins were stored at 4 °C at
concentrations of 0.5-1.5 mg of protein/ml. The His-tagged proteins
tended to precipitate out of solution; precipitation occurred more
readily when protein concentrations were higher. His-G6PD retained
activity for 2-3 weeks when stored at 4 °C in elution buffer (20 mM Tris-HCl, 500 mM NaCl, 1 M
imidazole, pH 7.8). His-OpcA rapidly lost its ability to activate G6PD
when it was stored in elution buffer; consequently, it was dialyzed in
several changes of 50 mM Tris maleate buffer (pH 6.5)
containing 10 mM MgCl2 prior to storage at
4 °C for 2-3 weeks. Neither protein was stable at
20 °C when
stored in buffers containing 0-50% glycerol nor under any condition
if diluted to less than 0.5 mg/ml. Losses of activity during
purification and storage have been reported previously for G6PD (4, 8,
18, 19).
-rabbit secondary antibody and a
p-iodophenol/Luminol chemiluminescent protocol (22) or with
alkaline phosphatase-conjugated goat
-rabbit secondary antibody
(1:50,000 dilution) and Amersham Pharmacia Biotech ECF substrate. When
the latter detection method was used, detection and quantitation of
chemifluorescent bands was performed using a Storm PhosphorImager with
ImageQuaNT software (Molecular Dynamics). Immunoprecipitations were
performed according to standard methods (21) with 100 µl of N. punctiforme extract (10-20 mg of protein/ml) and 5 µl of
-G6PD or
-OpcA antiserum or preimmune serum. After 1 h on
ice, 100 µl of 10% (v/v) protein A-Sepharose CL-4B beads was added,
and the mixture was incubated 1 h at 4 °C with gentle rocking.
The beads were washed four times with ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet
P-40). Electrophoresis of the immune complexes was performed using 10%
polyacrylamide SDS-PAGE gels with
-mercaptoethanol omitted from the
sample buffer. Immunoprecipitated proteins were detected by Western blotting.
-amylase (200 kDa), apoferritin (450 kDa), urease (545 kDa), and thyroglobulin (669 kDa). The gels were stained with Coomassie
Blue or G6PD activity stain (4) to identify standards and unknowns,
respectively. Average RF values for each protein at
each gel concentration were determined by measuring the distances from
the center of the bands to the dye front on duplicate gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-G6PD or
-OpcA antibodies. The G6PD activity clearly varied with growth conditions. In the experiment shown in Fig.
1, the activity of cells grown with
ammonium in the light was 0.035 µmol·min
1·mg of
protein
1. G6PD activity increased by 1.6-fold in
nitrogen-fixing cells grown in the light (0.055 µmol·min
1·mg of protein
1) and by
7.6-fold (0.265 µmol·min
1·mg of
protein
1) in nitrogen-fixing cells grown
heterotrophically in the dark with fructose. The increases in G6PD
activity were accompanied by increased synthesis of G6PD and OpcA, but
the aggregation states of G6PD and OpcA were unchanged (Fig. 1).
View larger version (27K):
[in a new window]
Fig. 1.
Western blot analysis of the aggregation
states and quantities of G6PD and OpcA in cell extracts prepared from
N. punctiforme cultures grown under different
conditions. Cell extracts were prepared from cultures grown
photoautotrophically with ammonium (lanes 1 and
4), photoautotrophically with dinitrogen (lanes 2 and 5), or heterotrophically in the dark with glucose and
dinitrogen (lanes 3 and 6) as described under
"Experimental Procedures." Aliquots of the cell extracts containing
70 µg of total protein were loaded on a 10% acrylamide gel, and the
proteins were separated by native PAGE. Lanes 1-3 of the
blot prepared from this gel were incubated with -G6PD antibody,
whereas lanes 4-6 were incubated with
-OpcA antibody.
The G6PD activities of the extracts in lanes 1,
2, and 3 were 0.035, 0.055, and 0.265 µmol of
NADP reduced·min
1·mg of protein
1,
respectively. The positions of G6PD, OpcA, and the native PAGE protein
standards are indicated.
-G6PD or
-OpcA antibodies. Each protein was immunoprecipitated with its
respective antibody, but the two proteins did not coimmunoprecipitate, and neither cross-immunoprecipitation nor immunoprecipitation with
pre-immune serum occurred (data not shown).
View larger version (55K):
[in a new window]
Fig. 2.
Migration of cyanobacterial G6PDs on a native
PAGE gel stained for G6PD activity. Native PAGE (7% acrylamide
gel) was performed with purified N. punctiforme His-G6PD and
with cell extracts prepared from N. punctiforme and
Anabaena 7120 cultures grown photoautotrophically with
ammonium to estimate the molecular mass and aggregation state of G6PD.
The gel was stained for G6PD activity as described under
"Experimental Procedures." One major band was detected in all
samples. Lane 1, Anabaena 7120 extract (83 µg
of protein); lane 2, N. punctiforme extract (168 µg of protein); lane 3, purified N. punctiforme
His-G6PD synthesized in E. coli (0.6 µg of protein). The
positions of the native PAGE protein standards are indicated.
View larger version (21K):
[in a new window]
Fig. 3.
Determination of the molecular mass of
N. punctiforme G6PD and His-G6PD by Ferguson
analysis. Native PAGE was performed using a series of gels (5, 6, and 7% acrylamide). Purified N. punctiforme His-G6PD and
cell extracts prepared from N. punctiforme and
Anabaena 7120 cultures grown photoautotrophically with
ammonium were loaded on each gel along with native PAGE standards.
After electrophoresis, lanes containing standards were stained with
Coomassie Blue, whereas the remaining lanes were stained for G6PD
activity. A, RF values were determined by
measuring the distances from the center of the bands to the dye front
on duplicate gels. Retardation coefficients for each standard and
unknown protein were then determined by plotting 100 log(RF × 100) as a function of the percent gel
concentration. The negative slope of each line is the retardation
coefficient. Standards: open triangles, bovine serum albumin
monomer (66 kDa); inverted open triangles, bovine serum
albumin dimer (132 kDa); open circles, -amylase (200 kDa); open squares, apoferritin (450 kDa); open
diamonds (obscured by other data points), urease hexamer (545 kDa); crosses, thyroglobulin (669 kDa). Unknowns:
closed squares, N. punctiforme extract G6PD;
closed circles, N. punctiforme His-G6PD;
closed triangles (some data points are obscured by other
data points), Anabaena 7120 extract G6PD. B, the
logarithm of the retardation coefficient for each protein standard was
next plotted against the logarithm of its molecular mass. The resulting
standard curve (closed diamonds) was used to determine the
molecular masses of the cyanobacterial G6PDs. Solid line,
N. punctiforme extract G6PD, 200 kDa; dotted
line, N. punctiforme His-G6PD, 229 kDa; dashed
line, Anabaena 7120 extract G6PD, 263 kDa.
Activity and aggregation state of G6PD in N. punctiforme strains
expressing different amounts of G6PD and OpcA protein
-G6PD and
-OpcA antibodies. G6PD activity was assayed using standard assay
conditions. G6PD activity is reported as mean ± S.E. with the
number of trials given in parentheses, and the units of G6PD activity
are µmol of NADP reduced · min
1 · mg
protein
1. The aggregation state of G6PD was determined for
each strain by comparing the migration of its G6PD to that of wild-type
N. punctiforme and to protein standards using native PAGE
and Western blotting.
1·mg of
protein
1 in the absence of strain UCD 466 extract to
0.127 µmol·min
1·mg of protein
1 when
strain UCD 466 extract was saturating (Fig. 4 and Table III). The increase in G6PD activity upon
the addition of strain UCD 466 extract occurred within 30 s after
the mixing of the extracts and remained constant after incubation of
the mixture on ice for more than 1 h. There was no increase in
G6PD activity when strain UCD 341 extract (no G6PD or OpcA) was added
to strain UCD 351 extract, nor was there any change in the G6PD
activity of wild-type N. punctiforme extract when extract
from strain UCD 466 was added (data not shown). Quantitative Western
blotting indicated that the strain UCD 351 extract used in the
experiment shown in Fig. 4 contained 10.3 ng of G6PD/µg of total
protein, whereas the strain UCD 466 extract contained 179 ng of
OpcA/µg of total protein. Therefore, the concentration of G6PD in the
assay was 13.0 nM, and the concentration of OpcA was varied
between 0 and 360 nM. When the data were fitted to the Hill
equation and plotted (Fig. 4, inset), the OpcA concentration
at half-maximal velocity (S0.5) was determined
to be 11.0 nM. Additional experiments performed with G6PD
and OpcA from different sources are summarized in Table III. Although
the concentrations of UCD 351 G6PD and His-G6PD used in these
experiments were similar, the concentration of OpcA required for
half-maximal activation was lower for UCD 351 extract G6PD than for
His-G6PD.
View larger version (23K):
[in a new window]
Fig. 4.
Enhancement of the G6PD activity of strain
UCD 351 extract upon the addition of UCD 466 extract. A fixed
amount of strain UCD 351 extract (73 µg of total protein) was mixed
with varying amounts of strain UCD 466 extract and assayed for G6PD
activity as described under "Experimental Procedures." Assay
mixtures contained 2 mM NADP and 5 mM G6P. The
quantities of G6PD and OpcA present in the strain UCD 351 and UCD 466 extracts, respectively, were determined by quantitative Western
blotting (not shown), which indicated that the assays were performed
with 13 nM strain UCD 351 G6PD and 0-360 nM
strain UCD 466 OpcA. Kinetic parameters were obtained by fitting the
data to the Hill equation and are as follows:
Vmax = 0.127 µmol·min 1·mg
strain UCD 351 protein, S0.5 = 11.0 nM, and n = 1.5. Inset shows a
Hill plot of the data.
Determination of the OpcA concentration required for half-maximal
activation of G6PD from different sources
1 · mg UCD 351 extract or
His-G6PD
1, and activity is reported as mean ± S.E. with
the number of trials given in parentheses. Values of n, the
Hill coefficient, and S0.5, the concentration of
OpcA required for half-maximal activation of G6PD, were determined by
fitting the data to the Hill equation.
-OpcA antibodies were used to examine the interaction between G6PD
and OpcA. Aliquots (40 µl) of
-OpcA or preimmune serum were added
either to His-OpcA (4.4 µg, 80 pmol) alone or to a mixture containing
an identical amount of His-OpcA plus His-G6PD (0.5 µg, 8 pmol). The
samples were incubated on ice for 1 h. The serum-treated His-OpcA
samples were then mixed with His-G6PD (0.5 µg, 8 pmol) to test their
ability to enhance G6PD activity, whereas the serum-treated samples
containing the mixture of His-OpcA and His-G6PD were
assayed directly for G6PD activity. His-OpcA that had been
incubated with
-OpcA retained only 3% of its initial ability to
activate His-G6PD; no loss in the ability to activate G6PD was
observed when His-OpcA was incubated with preimmune
serum. Inactivation of His-OpcA also occurred when it was
mixed with His-G6PD prior to the addition of
-OpcA; only 15% of the
initial activity of the His-G6PD plus His-OpcA protein mixture remained after incubation with
-OpcA, whereas 87% of the initial activity of
the mixture remained after incubation with preimmune serum.
View larger version (17K):
[in a new window]
Fig. 5.
The affinity of N. punctiforme
G6PD for G6P substrate was increased in the presence of
OpcA. A, activity versus G6P substrate
concentration curves for G6PD in strain UCD 351 extract (open
circles) or wild-type N. punctiforme extract
(closed circles). Strain UCD 351 overproduces G6PD but lacks
OpcA, whereas wild-type N. punctiforme contains
approximately equal amounts of both proteins. Here the amount of each
extract was held constant at 70 µg of total protein, NADP was held
constant at 2 mM, and the G6P concentration was varied.
Because strain UCD 351 contains more G6PD per unit total protein than
wild-type N. punctiforme, G6PD activity for each strain is
expressed as a percentage of the maximum activity obtained for that
strain. B, activity versus G6P substrate
concentration curves for purified His-G6PD in the absence (open
squares) or presence (closed squares) of saturating
His-OpcA. His-G6PD (0.5 µg; 8 pmol) was assayed alone or was mixed
with His-OpcA (4.4 µg; 80 pmol). The NADP concentration was held
constant at 2 mM. Kinetic parameters were determined by
fitting the data to the Hill equation and are
Vmax = 80.4 µmol·min 1·mg
His-G6PD, S0.5 = 58.9 mM, and
n = 2.4 for His-G6PD in the absence of His-OpcA and
Vmax = 77.7 µmol·min
1·mg
His-G6PD, S0.5 = 1.8 mM, and
n = 0.9 for His-G6PD in the presence of His-OpcA.
Inset shows detail of the region between 0 and 10 mM G6P.
Km(app) values for N. punctiforme G6PD and for strain UCD 351 G6PD and His-G6PD assayed in the absence and presence of His-OpcA
View larger version (20K):
[in a new window]
Fig. 6.
Reversible redox modulation of the activity
of His-G6PD occurred in the presence of His-OpcA and thioredoxin.
Dithiothreitol (DTT)-reduced thioredoxin (1 mM
Spirulina thioredoxin plus 2 mM DTT) was added
to His-OpcA at time 0. The initial mixture contained 3.2 µM His-OpcA, 32 µM thioredoxin, and 63 µM DTT. After 1 min, and at 3-min intervals thereafter,
aliquots containing 160 pmol of His-OpcA and 1600 pmol of thioredoxin
were removed, mixed with 16 pmol of His-G6PD and assayed immediately
for G6PD activity (closed squares, solid lines).
At 6-min intervals, indicated by arrows, sodium
tetrathionate (STT) or DTT was added to the His-OpcA plus thioredoxin
mixture to the final concentration indicated to change the redox state
of the thioredoxin. The control experiment (open squares,
dotted line) was performed in an identical manner except
that buffer replaced thioredoxin (but not DTT) in the initial mixture.
In the absence of any His-OpcA or thioredoxin, the activity of His-G6PD
was 0.945 µmol·min 1·mg of protein
1.
In the presence of His-OpcA and absence of thioredoxin, the activity of
His-G6PD increased to 14.4 µmol·min
1·mg of
protein
1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 mg/ml) and in a
"low activity" form when it was diluted. Both forms of the enzyme
exhibited sigmoidal kinetics, but the apparent Km (S0.5) for G6P at pH 7 to 8 was 2.2-7.2
mM for the concentrated enzyme and 49-54 mM
for the dilute enzyme. OpcA was most likely absent from this purified
Anabaena 7120 G6PD preparation: A typical specific activity
of 2-6 µmol·min
1·mg of protein
1 was
reported for the Anabaena 7120 enzyme, when it was assayed at 20 mM G6P (8). This value is slightly higher than the
average specific activity of 0.9 ± 0.1 µmol·min
1·mg of protein
1 we typically
obtained for purified His-G6PD preparations at 5 mM G6P,
and is comparable to the specific activity of 8.0 µmol·min
1·mg of protein
1 we observed
for His-G6PD at 20 mM G6P (Fig. 5B). It is more
than an order of magnitude lower than the highest specific activity (86 µmol·min
1·mg His-G6PD protein
1 at 5 mM G6P) we obtained for the OpcA-activated enzyme (Table III). From Western blots, microscopic measurements, and protein and Chl
a assays we have determined that the average wild-type N. punctiforme cell is 2.5 µm x 4 µm and contains ~1
mg of G6PD monomer and 20 mg of Chl a per g of total protein
when cultured with ammonium. Assuming the cell is a cylinder, we
calculate a cell volume of 2 × 10
11 ml, and if a
cell contains 1.75 × 10
13 g of Chl a
(14), we estimate that the cellular G6PD concentration is about 0.4 mg/ml. If G6PD is present at a similar concentration in
Anabaena 7120 cultured with nitrate, at 2 mg/ml or greater, purified "high activity" G6PD could be as much as 5 times more concentrated in vitro than it is in the cell. Purified
cyanobacterial G6PD has the unusual in vitro properties of
inactivation and precipitation/activation in dilute and concentrated
suspension, respectively (see "Experimental Procedures" and ref.
8). We speculate that nonphysiologically high concentrations of the
enzyme, or of effectors such as G6P or NADP, and physicochemical
factors such as low pH, might stabilize purified G6PD in the absence of
OpcA, keeping it in an activated or partially activated conformation
that resembles the conformation that is characteristically found in the
cell when OpcA is present.
-OpcA antibody indicated that repeated interactions
with His-OpcA were necessary to keep His-G6PD in an activated state. If
activation required only a single interaction between the two proteins,
as would be expected if OpcA activated G6PD by oxidation, then addition
of
-OpcA after activation had occurred should have had no effect on
G6PD activity. The decline in G6PD activity we observed after the
addition of
-OpcA to the His-G6PD plus His-OpcA mixture implies that
further interaction between the two proteins was required to keep the
G6PD in an activated state and that binding of
-OpcA to His-OpcA
either physically prevented this interaction or rendered the His-OpcA inactive.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. L. Ingraham and I. H. Segel for helpful advice and discussions and E. L. Campbell and F. C. Wong for critical reading of the manuscript.
![]() |
Note Added in Proof |
---|
The recent deposition of the Corynebacterium glutamicum opcA and zwf sequences in the GenBankTM database has brought to our attention the work of Moritz et al. (Moritz, B., Striegel, K., de Graaf, A. A., and Sahm, H. (2000) Eur. J. Biochem. 267, 3442-3452), who detected the OpcA protein in purified G6PD preparations from C. glutamicum. Outside of the division Cyanobacteria, sequences with similarity to OpcA have been identified only in members of the genera Corynebacterium, Streptomyces, Mycobacterium, and Deinococcus. Like the putative OpcA protein sequences from the other Gram-positive and the one Deinococcus group eubacteria, the C. glutamicum sequence has limited similarity (32%) to the N. punctiforme OpcA sequence and lacks the N-terminal and C-terminal amino acid sequences that are consistently present in the cyanobacterial OpcA proteins. Despite these differences, the copurification of C. glutamicum G6PD and OpcA implies that these smaller OpcA proteins may play a role in the activation of G6PD similar to that of the N. punctiforme OpcA.
![]() |
FOOTNOTES |
---|
* This work was supported by the U. S. National Science Foundation (Grant MCB 96-04270).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Section of
Microbiology, University of California, Davis, One Shields Ave., Davis, CA 95616. Tel.: 530-752-3346; Fax: 530-752-9014; E-mail: jcmeeks@ucdavis.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010472200
2 The nucleotide sequence of the N. punctiforme opc operon was previously deposited in the GenBank data base under GenBankTM accession number L32796.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: OPP, oxidative pentose phosphate; G6PD, glucose-6-phosphate dehydrogenase; G6P, glucose 6-phosphate; Anabaena 7120, Anabaena sp. strain PCC 7120; PAGE, polyacrylamide gel electrophoresis; Synechococcus 7942, Synechococcus sp. strain PCC 7942; MOPS, 3-(N-morpholino)propanesulfonic acid; Ap, ampicillin; Nm, neomycin; Chl a, chlorophyll a; PCR, polymerase chain reaction; His, histidine fusion tag; MBP, maltose binding protein fusion tag; DTT, dithiothreitol; STT, sodium tetrathionate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Winkenbach, F., and Wolk, C. P. (1973) Plant Physiol. 52, 480-483 |
2. | Apte, S. K., Rowell, P., and Stewart, W. D. P. (1978) Proc. R. Soc. Lond. B Biol. Sci. 200, 1-25 |
3. | Bothe, H., and Neuer, G. (1988) Methods Enzymol. 167, 496-501 |
4. | Schaeffer, F., and Stanier, R. Y. (1978) Arch. Microbiol. 116, 9-19[Medline] [Order article via Infotrieve] |
5. | Cossar, J. D., Rowell, P., and Stewart, W. D. P. (1984) J. Gen. Microbiol. 130, 991-998 |
6. | Buchanan, B. B. (1980) Annu. Rev. Plant. Physiol. 31, 341-374 |
7. | Udvardy, J., Borbely, G., Juhasz, A., and Farkas, G. L. (1984) J. Bacteriol. 157, 681-683[Medline] [Order article via Infotrieve] |
8. | Gleason, F. (1996) Arch. Biochem. Biophys. 334, 277-283[CrossRef][Medline] [Order article via Infotrieve] |
9. | Sundaram, S., Karakaya, H., Scanlan, D. J., and Mann, N. H. (1998) Microbiology 144, 1549-1556[Abstract] |
10. | Summers, M. L., Wallis, J. G., Campbell, E. L., and Meeks, J. C. (1995) J. Bacteriol. 177, 6184-6194[Abstract] |
11. | Summers, M. L., and Meeks, J. C. (1996) Mol. Microbiol. 22, 473-480[Medline] [Order article via Infotrieve] |
12. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
13. | Newman, J., Karakaya, H., Scanlan, D. J., and Mann, N. H. (1995) FEMS Lett. 133, 187-193 |
14. | Cohen, M. F., Wallis, J. G., Campbell, E. L., and Meeks, J. C. (1994) Microbiology 140, 3233-3240[Abstract] |
15. | Campbell, E. L., and Meeks, J. C. (1992) J. Gen. Microbiol. 138, 473-480 |
16. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
17. | Summers, M. L. (1995) The Role and Regulation of Glucose-6-Phosphate Dehydrogenase in Diazotrophic and Dark Heterotrophic Growth of the Cyanobacterium Nostoc sp, strain ATCC 29133Ph.D. dissertation , University of California, Davis |
18. | Levy, H. R. (1979) in Advances in Enzymology (Meister, A., ed) , pp. 97-192, John Wiley & Sons, NY |
19. |
Wenderoth, I.,
Scheibe, R.,
and von Schaewen, A.
(1997)
J. Biol. Chem.
272,
26985-26990 |
20. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology , John Wiley & Sons, NY |
21. | Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
22. | Hanson, T. E., Forchhammer, K., Tandeau de Marsac, N., and Meeks, J. C. (1998) Microbiology 144, 1537-1547[Abstract] |
23. | Udvardy, J., Juhasz, A., and Farkas, G. L. (1983) FEBS Lett. 152, 97-100[CrossRef][Medline] [Order article via Infotrieve] |
24. | Buchanan, B. B. (1991) Arch. Biochem. Biophys. 288, 1-9[Medline] [Order article via Infotrieve] |
25. | Scheibe, R. (1990) Bot. Acta 103, 327-334 |
26. | Wendt, U. K., Hauschild, R., Lange, C., Pietersma, M., Wenderoth, I., and von Schaewen, A. (1999) Plant Mol. Biol. 40, 487-494[CrossRef][Medline] [Order article via Infotrieve] |
27. | Scheibe, R., Geissler, A., and Fickenscher, K. (1989) Arch. Biochem. Biophys. 274, 290-297[Medline] [Order article via Infotrieve] |
28. | Black, T. A., Cai, Y., and Wolk, C. P. (1993) Mol. Microbiol. 9, 77-84[Medline] [Order article via Infotrieve] |
29. | Elhai, J., and Wolk, C. P. (1988) Gene 68, 119-138[CrossRef][Medline] [Order article via Infotrieve] |
30. | Rippka, R., and Herdman, M. (1992) Pasture Culture Collection of Cyanobacterial Strains in Axenic Culture , Institut Pasteur, Paris |