(Received for publication, November 10, 1995; and in revised form, January 2, 1996)
From the
Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.
Phosphoribulokinase (EC 2.7.1.19) catalyzes the final step in
the regeneration of ribulose 1,5-bisphosphate, the acceptor for
CO in photosynthetic carbon assimilation, from
3-phospho-D-glycerate. This kinase reaction, in which the
-phosphoryl of ATP is transferred to the C-1 hydroxyl of Ru-5-P, (
)provides the only avenue by which Calvin cycle
intermediates can be rechanneled for utilization in CO
fixation. As a means of coupling photosynthetic carbon reduction
with photosynthetic electron transport, the activity of PRK is
modulated by photon flux. In the case of prokaryotic photosynthetic
organisms, regulation of PRK (typically an octamer of 32-kDa subunits)
is mediated by the allosteric activator NADH, the intracellular
concentration of which is light-dependent (for a review, see (1) ). By contrast, regulation of eukaryotic PRK (typically a
dimer of 40-kDa subunits) (2, 3, 4) is
mediated by the redox active protein thioredoxin, which participates in
sulfhydryl/disulfide exchange reactions(5, 6) . In the
light, thioredoxin is maintained in its reduced form by the electron
carrier ferredoxin. Reduced thioredoxin then reduces the regulatory
disulfide of the inactive form of PRK to free sulfhydryls, thereby
generating the active form of PRK.
In addition to the striking
differences in molecular architecture and mode of regulation between
prokaryotic and eukaryotic PRK, their catalytic parameters are also
quite distinct. As an example, the k of plant
PRK is about 10-fold higher than that of the bacterial
counterpart(1) . Hence, the two classes of PRK must be
considered as separate entities, each deserving of independent
scrutiny. Structure-function studies of the bacterial (Rhodobacter
sphaeroides) PRK have been greatly facilitated by the recent
redesign of an expression plasmid that overproduces the recombinant
enzyme in Escherichia coli to the extent of about 25% of the
total soluble protein(7, 8) . Although the gene for
spinach PRK has also been cloned (9, 10) and expressed
in E. coli(11, 12) , the yield of recombinant
enzyme is very low (
0.5% of total soluble protein) due to the dual
problems of susceptibility to proteolysis and inherent toxicity of
spinach PRK to the host.
To circumvent these impediments, we have explored utilization of the yeast Pichia pastoris as an expression host for spinach PRK. In this paper, we report our success in this endeavor, including efficient purification of the recombinant enzyme and its characterization to validate authenticity to PRK isolated from spinach. We also describe construction, purification, and characterization of site-directed mutants of spinach PRK, in which the regulatory cysteinyl residues are replaced both individually and in combination so as to gain understanding of the molecular basis of oxidative deactivation.
Figure 1:
Integration of the PRK expression cassette into P. pastoris chromosomal DNA. A, schematic representation of the displacement of the AOX1 sequence of GS115 by the expression cassette excised from
pFL451 by NotI digestion. The displacement product, denoted aox1::PRK, lacks AOX1 and contains PRK-HIS4. The sequences flanking the PRK-HIS4 segment
are homologous to the target sequences flanking AOX1. The 5`
and 3` arrows denote the locations of sequencing/PCR primers. B and C, Southern transfer hybridization of NotI-BglII-digested plasmid and BglII-digested genomic DNAs. In B, AOX1 is
used as the probe. The coding sequence of AOX2 is nearly
identical to AOX1 and is thus also visualized. In C, PRK is used as the probe. D, ethidium bromide-stained
agarose gel of PCR-amplified intact plasmid and bulk genomic DNAs. The
identity of the amplified DNA was confirmed by dideoxy-terminator cycle
sequencing (not shown). Note in B and D the absence
of AOX1 in
aox1::PRK and in C that the
entire disruption cassette is incorporated into chromosome
aox1::PRK.
All mutant PRKs were purified by the same protocol and displayed chromatographic behavior indistinguishable from that of wild-type.
The concentration of PRK was determined at 280 nm based on an extinction coefficient of 7.17 for a 1% (10 mg/ml) solution in a 1-cm light path(13) .
For each construction, crude extracts were
prepared from 10-12 methanol-induced his methanol
isolates. These extracts were examined
for PRK production by Western blotting with a polyclonal anti-PRK
antiserum, and the most prolific producer of PRK was selected. These
PRK-positive transformants were further characterized by Southern
transfer hybridization, PCR amplification of the AOX1 region
and DNA sequencing of the amplified segment (Fig. 1). Isolates
chosen for production of PRK were devoid of AOX1 sequences and
exhibited strong PRK signals. In the methanol-induced
cultures, PRK reached 4-6% of the total soluble protein at
60-72 h post-induction. Longer term incubation did not increase
the specific activity, and total protein levels declined.
SDS-polyacrylamide gel electrophoresis and Western blot analysis of a
large scale culture of the wild-type transformant is shown in Fig. 2. Note the absence of significant degradation of the PRK
polypeptide chain. The diffuse leading edge of the PRK band detected by
Western blotting is apparent even in the enzyme isolated from spinach
and hence does not reflect an anomaly unique to the recombinant kinase.
Figure 2:
Synthesis of PRK following
methanol-induction of transformed P. pastoris. In both panels,
lane identifications are as follows: lane 1, purified spinach
PRK (specific activity of 500 units/mg); lane 2, extract from
untransformed GS115 (specific activity of 0 units/mg); lane 3,
uninduced aox1::PRK extract (specific activity of 0.7
unit/mg); lane 4,
aox1::PRK extract 24 h
post-induction (specific activity of 11 units/mg); lane 5, 48
h post-induction (specific activity of 12 units/mg); lane 6,
72 h post-induction (specific activity of 19 units/mg). A,
SDS-polyacrylamide gel electropherogram stained with Coomassie Blue; lane 1, 500 ng of purified spinach PRK; lanes
2-6, 10 µg of total protein. B, Western
transfer of an electropherogram probed with anti-PRK; lane 1,
50 ng of purified spinach PRK; lanes 2-6, 1 µg of
total protein.
Figure 3: Polyacrylamide gel electrophoresis of PRKs. A, Coomassie-Blue-stained nondenaturing gel with all samples at 1 µg/lane. B, silver-stained denaturing gel with all samples at 50 ng/lane. Lanes 1 and 8, molecular markers (Novex, Mark 12); lane 2, authentic spinach PRK; lane 3, wild-type recombinant; lane 4, C16S; lane 5, C55S; lane 6, C16S-C55S; lane 7, C16S-C55S-C244S-C250S.
Figure 4:
Inactivation of recombinant PRKs by DTNB.
Reaction mixtures contained PRK (10 µM subunits) in 50
mM Bicine (pH 8.0), 20% (v/v) glycerol, and inactivations were
initiated by addition of DTNB to a final concentration of 50
µM. Periodically, aliquots were removed and assayed as
described under ``Experimental Procedures.'' Samples depicted
are as follows: wild-type (), C55S (
), C16S (
),
C16S-C55S (
), C16S-C55S-C244S-C250S (
), controls of
wild-type and mutant proteins lacking DTNB
(
).
The electrophoretic
mobilities of the four purified mutant proteins are similar to those of
wild-type PRK (Fig. 3). However, each of the three mutants
carrying a seryl substitution at position 16 migrates as a poorly
resolved doublet under nondenaturing conditions. This
microheterogeneity does not appear to be due to contamination by
another protein, because the specific activity of C16S approximates
that of the wild-type enzyme. Kinetic analyses of the mutant proteins
are summarized in Table 2. In reasonable agreement with our
earlier report(11) , particularly in view of crude preparations
used at that time, C16S is almost fully active, and C55S displays only
about 20% of wild-type activity. The activity of the C16S-C55S double
mutant is decreased another 2-fold (10% of wild-type), but the
quadruple mutant altogether lacking cysteinyl residues is not further
impaired. Although none of the cysteinyl substitutions dramatically
impact the K for ATP (e.g. the same value
was obtained for the quadruple mutant and wild-type enzyme), all three
mutants with replacements for Cys-55 have substantially elevated K
values for Ru-5-P. These three mutants, in
contrast to C16S and wild-type enzyme, are subject to inhibition by ATP
at high concentrations in a manner adequately modeled by uncompetitive
substrate inhibition.
One set of V and K
values for C55S, C16S-C55S, and
C16S-C55S-C244S-C250S presented in Table 2are based on velocity
measurements at concentrations of Ru-5-P not exceeding 4 mM and fitting the data to a hyperbolic response. However, distinct
deviation from hyperbolic kinetics is observed at higher concentrations
of Ru-5-P, and saturation is not achieved even at 17 mM Ru-5-P (Fig. 5). Inclusion of sulfate (35 mM) in the assay
mixture results in clear-cut hyperbolic kinetics and apparent
enhancement of kinase activity at any given concentration of Ru-5-P.
Hence, the second set of kinetic parameters presented in Table 2are those determined in the presence of sulfate. The
stimulatory effect of sulfate on the three mutants lacking Cys-55
appears to be ion-selective, as chloride is ineffective and phosphate
is inhibitory (Fig. 6). Sulfate does not enhance the activity of
either the wild-type enzyme or the C16S mutant.
Figure 5: Effect of sulfate on the Ru-5-P-concentration dependence of kinase activity of position 55 mutants. Initial velocities in the presence and absence of 35 mM sodium sulfate are shown. As the relative responses for C55S, C16S-C55S, and C16S-C55S-C244S-C250S were the same, single curves are displayed for the two conditions. See ``Experimental Procedures'' for further details of assays.
Figure 6: Effect of anions on the relative activity of position 55 mutants. In all cases, the Ru-5-P and ATP concentrations were each 1 mM. As the relative responses for C55S, C16S-C55S, and C16S-C55S-C244S-C250S were the same, single curves are displayed for each anion. Error bars denote standard deviation based on averages of data derived from all three position 55 mutants. See ``Experimental Procedures'' for further details of assays.
The double and quadruple mutants (each of which lacks both regulatory sulfhydryls) are impervious to DTNB, while C55S and C16S (each of which retain one regulatory sulfhydryl) display wild-type and diminished sensitivity to DTNB, respectively (Fig. 4). Whereas 2 molar eq of 5-thio-2-nitrobenzoate are formed/mol of wild-type PRK subunit inactivated by DTNB (reflective of intrasubunit disulfide bond formation)(14) , only 1 molar eq of the chromophore is formed during inactivation of the C55S and C16S mutants (reflective of derivatization of the available sulfhydryl group by the reagent moiety) (data not shown). As in the case of the wild-type enzyme, the inactivations of C55S and C16S by DTNB are readily reversed by DTT (data not shown).
PRK is one of several chloroplast enzymes to be regulated by
thioredoxin (for reviews, see (6) and (23) ). Others
include NADP-dependent malate dehydrogenase,
fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and
glyceraldehyde-3-phosphate dehydrogenase. Among these enzymes, PRK is
the only one in which the regulatory sulfhydryls (Cys-16 and Cys-55)
are located at the active site(6, 14) . Hence, the
question naturally arises as to whether the complete loss of kinase
activity that accompanies disulfide bond formation between Cys-16 and
Cys-55 is due to removal of catalytically important sulfhydryl groups
or to a conformational change. Prior studies have indicated that both
factors contribute to the oxidation-associated deactivation. Analysis
of site-directed mutants with single amino acid substitutions for
either of the regulatory cysteinyl residues showed that Cys-55 enhances k by 10-fold and that Cys-16 is inconsequential
to catalysis(11, 12) . Localized conformational
differences between the oxidized and reduced form of the enzyme were
invoked by the observed efficient intrasubunit cross-linking of the
regulatory sulfhydryls by bifunctional reagents spanning distances as
short as 3.5 Å and as long as 9 Å(24) . Thus, a
part of the deactivation concomitant with oxidation could be attributed
to restricted conformational flexibility imposed by the disulfide bond.
We wished to evaluate further the basis of oxidative deactivation of PRK, because the catalytic consequences of simultaneous replacement of both regulatory sulfhydryls were not explored in earlier mutagenesis studies. Given the paucity of recombinant spinach PRK elaborated by reported E. coli expression systems, we sought a superior host not only to advance the present inquiry but to also facilitate future studies of the structure and function of the kinase.
Our original expression cassette for spinach PRK relied on a tac promoter in concert with a ribosomal binding site to maximize production of the mature form of the enzyme as found in the chloroplast(9, 11) . This promoter-ribosomal binding site unit had been used by our laboratory to express recombinant ribulose-bisphosphate carboxylase/oxygenase in E. coli at levels approaching 20% of the total soluble protein(25) . Levels of PRK expression, however, rarely surpassed 0.5% and frequently were only 0.2% of extractable protein. Even these meager levels of PRK production required cultures to be grown at or below 30 °C in the presence of arabinose, which suppressed the susceptibility of the recombinant enzyme to proteolysis. The arabinose effect could be duplicated in an arabinose-constitutive host without the addition of arabinose to the culture, suggesting that an inducible protein, rather than the sugar or its metabolites, was responsible for the protection. Western blots of crude extracts indicated some degradation, even under ``optimal'' conditions. Mutant PRKs, severely deficient in catalytic activity, were also poorly expressed, suggesting that the recombinant protein itself was eliciting a stress response. Invariably, induction of transcription of the wild-type or mutant genes encoding PRK led to cessation of cell division followed by cell death.
The
PRK coding sequence, like many of the spinach genome, utilizes AGA or
AGG codons for arginine predominantly (for 13 out of the 14 total
arginyl residues), whereas the preferred arginine codons in E. coli are CGN. Arg-23 and Arg-24 of PRK are encoded by AGG; this
presence of a tandem of rare codons early in the transcript may lead to
ribosome pausing that is deleterious to high levels of expression and
may further compromise the host by depleting charged pools of the rare
tRNA(26) . Furthermore, the sequence AGGAGG resembles a
ribosomal binding site and may lead to unproductive internal
initiations on the transcript(27) . In efforts to overcome
these potential problems, a clone of the arginyl-tRNA gene, dnaY, was co-expressed with PRK, and codons 23 and
24 were mutated to CGC codons. Neither of these measures, individually
or in combination, enhanced PRK expression.
Aside from the tac promoter, several other highly regulated promoters were tried in
alternative PRK expression cassettes. Neither the promoter of the trp operon (inducible with indole-acrylic acid), the
P
promoter (inducible by heat shock in a cI857
host), nor a T7 phage promoter (controlled by induction of T7 RNA
polymerase) supported PRK production above 0.2-0.3% of soluble
protein.
In view of our inability to overcome these impediments, we turned our attention to eukaryotic host organisms. One system investigated was the GAL1 promoter of Saccharomyces cerevisiae, which is highly induced by galactose. A GAL1 promoter PRK expression cassette was constructed and introduced into the host yeast on a high copy episomal vector. Upon induction with galactose, the level of PRK produced was still only 0.2-0.3% of soluble protein. We were encouraged, however, to note very little degradation of the recombinant enzyme based on Western blots of the crude extract and were thus prompted to evaluate the alcohol oxidase promoter (AOX1) of the methylotrophic yeast P. pastoris as an avenue for obtaining recombinant PRK. Recently, a number of proteins that had proven difficult to produce in standard hosts have been obtained in high yields by use of this system(28) . Similarly, when induced with methanol, the transformant of P. pastoris described herein synthesizes large amounts of PRK (about 5% of total soluble protein), and the synthesis is not compromised by proteolytic degradation. Efficient expression, coupled with facile isolation, enables us to recover about 25 mg of purified PRK (either wild-type or mutants) from 1 liter of cell culture.
In corroboration
of our earlier findings, Cys-16 of PRK does not play any role in
catalysis. The V of the C16S mutant equals that
of the wild-type enzyme, and the very slight increase of the K
for ATP with the C16S mutant is not surprising,
given the location of Cys-16 within the binding domain for
ATP(12, 13, 29, 30) . In both our
prior study (11) and an independent one(12) , the C55S
mutant was reported to have a V
of 10% of the
wild-type value. We now observe a value about twice this large with the
same mutant constructed in the P. pastoris system. Greater
credence can be placed in the present kinetic analyses, because the use
of purified proteins eliminates the margin of error introduced by
reliance on Western blotting to quantify kinase concentrations in the
impure preparations analyzed before. Thus, Cys-55 would still be
classified as catalytically facilitative, but its contribution to rate
enhancement is maximally 5-fold. This contribution drops to only
2.5-fold if the stimulatory effect of sulfate is taken into account.
The substantially increased K
for Ru-5-P, with the
C55S mutant relative to the wild-type enzyme, is consistent with the
assignment of Cys-55 to the binding domain for Ru-5-P(31) .
The C16S-C55S double mutant serves as a better gauge of the combined catalytic contribution of the two regulatory sulfhydryls than the single mutants assessed individually. Barring synergistic effects, insertion of a seryl substitution at position 16 of the C55S mutant would be catalytically negligible. However, the double mutant is only 50% (in the absence of sulfate) or 70% (in the presence of sulfate) as active as C55S, thereby revealing synergism between the two sites of substitution. Even so, the complete inactivation of PRK concomitant with oxidation to the disulfide form cannot be accounted for merely by the absence of free sulfhydryls. The significant catalytic competency retained by the double mutant is consistent with our prior contention that conformational constraints imposed by the disulfide bond contribute to oxidative deactivation of PRK(24) . Retention of activity by the quadruple mutant, devoid of cysteinyl residues, underscores the modest collective contribution of sulfhydryl groups to catalytic turnover.
We assume that the substantial stimulation of kinase activity of the position 55 mutants by sulfate reflects conformational differences between these three mutants and the wild-type enzyme and that sulfate favors the wild-type conformation. Although we have not characterized this phenomenon in detail, we note that sulfate activation of phosphoglycerate kinase has been thoroughly analyzed. In that case, activation is clearly mediated by a sulfate-induced conformational change(32, 33, 34, 35) .
In summary, we have designed the first efficient expression system for a thioredoxin-regulated PRK. We have proceeded to exploit this system via site-directed mutagenesis in order to clarify both the relevance of sulfhydryl groups to kinase activity and the basis of oxidation-associated, regulatory deactivation of PRK.