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INTRODUCTION |
Ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco1; EC 4.1.1.39)
catalyzes the covalent addition of CO2 to ribulose 1,5-bisphosphate producing two molecules of 3-phosphoglycerate (reviewed in Ref. 1). This reaction is the first step in the fixation
of atmospheric CO2 into the food chain. A competing side reaction occurs in the presence of oxygen, leading to one molecule of
3-phosphoglycerate and one molecule of 2-phosphoglycolate.
Rubisco is found predominantly in higher plants, algae, cyanobacteria,
and other photosynthetic organisms (reviewed in Ref. 2). In most of
these organisms, Rubisco is a hexadecamer consisting of eight large (L)
and eight small (S) subunits in an L8S8
arrangement (type I or form I). Another type of Rubisco, found in
some photosynthetic
-purple bacteria like Rhodospirillum
rubrum, is a homodimer of L subunits only (type II or form II)
(3). The nonphotosynthetic chemoautotrophic
-purple bacterium
Thiobacillus denitrificans also harbors a type II Rubisco,
along with a distinct type I Rubisco (4). A new classification of
Rubisco enzymes, based on their primary structure relatedness, has also
recently been proposed by Tabita (2). A detailed phylogenetic analysis
of Rubiscos has also been conducted (5), and from these studies, type I enzymes can first be divided into two major branches, the
"Green-like" and "Red-like" Rubiscos. The two groups can be
further divided into two subgroups, respectively. Type II Rubiscos form
a uniform class of enzymes including those from R. rubrum,
Rhodobacter sphaeroides, and T. denitrificans. The incongruency of Rubisco phylogenies with those
derived from 16 S rRNA sequences (6), or other macromolecules, has
suggested the occurrence of horizontal gene transfer during the
evolution of Rubisco and the organisms that harbor the enzyme (5).
Concerning archaea, the Rubisco from halobacterium Haloferax
mediterranei is the only enzyme that has been characterized (7). H. mediterranei Rubisco is composed of L and S subunits and,
from this viewpoint, corresponds to type I Rubisco. Isolation of the genes encoding the subunits has not been characterized as yet, and
therefore the primary structures have not been determined. Although
there have been no reports of a Rubisco from a hyperthermophilic archaeon, putative Rubisco genes have been reported through genome analysis of the archaea Methanococcus jannaschii (8) and
Archaeoglobus fulgidus (9). The deduced amino acid sequences
indicate that they do not belong to the type I and type II Rubiscos.
However, the protein products of the genes have not been identified nor characterized, and evidence that they actually harbor Rubisco activity
is an urgent subject of Rubisco research (5).
Pyrococcus kodakaraensis KOD1 is a hyperthermophilic
archaeon that was isolated from a solfatara at a wharf of Kodakara
Island, Kagoshima, Japan (10). P. kodakaraensis KOD1 shows
optimum growth at 95° C, and enzymes characterized from the strain
have all shown extreme thermostability. We have been conducting genome
analysis and have identified and characterized genes such as those
encoding chaperonin (11), DNA polymerase (12), ribose phosphate
pyrophosphokinase (13), DNA recombination protein (14), and
indolepyruvate ferredoxin oxidoreductase (15). Many of the proteins
from P. kodakaraensis KOD1 show distinct structures and
characteristics compared with proteins from eukaryotes and bacteria
(11-15). Through sequence analysis of an
AscI-AscI fragment (200 kilobase pairs) of the genome, we found an open reading frame that displayed similarity with
previously reported Rubisco genes. Here we report that the gene
(Pk-rbc1) actually encodes an extremely thermostable and highly carboxylase specific Rubisco and that the primary structure is
distinct from type I and type II Rubiscos.
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MATERIALS AND METHODS |
Strains, Plasmids, and Media--
P. kodakaraensis
KOD1 was cultivated using a medium described in Ref. 10 with additional
saturation of carbon dioxide by bubbling with dry ice.
Escherichia coli JM109 and the vector pUC19 were used for
cloning and gene manipulation. E. coli BL21(DE3) (Stratagene, La Jolla, CA) and the vector pET21a(+) (Novagen Inc., Madison, WI) were used for overexpression of Pk-rbc1.
Pk-rbc1 was originally identified on a DNA fragment inserted into
EMBL4 phage vector. LB medium was used for cultivation of E. coli and NZYM medium for amplification of phage (16).
DNA Manipulation and Sequencing--
For isolation of plasmid
DNA, Plasmid Mini-, Midi- and Lambda Kits (Qiagen, Hilden, Germany)
were used along with the alkaline extraction method (16). Restriction
enzymes and modifying enzymes were purchased from Toyobo (Osaka,
Japan), Takara (Kyoto, Japan), and Boehringer Mannheim (Mannheim,
Germany). DNA sequencing on both strands of DNA was conducted using the
ABI PRISM kit and Model 310 capillary DNA sequencer (Perkin-Elmer).
Sequence Alignment--
The multiple alignment of protein
sequences and the number of similarity between sequences were obtained
with the program ALIGN contained within the ClustalW program provided
by DNA Data Bank Japan (DDBJ).
Gene Expression and Protein Purification--
After insertion of
the Pk-rbc1 into pET21a(+), the plasmid was transformed into
E. coli BL21(DE3). Transformants were cultivated until
optical density at 660 nm reached 0.5. Isopropyl-D-thiogalactopyranoside (0.1 mM) was
added to induce gene expression for 4 h, when cells were harvested
and disrupted by sonication. After centrifugation (7,500 × g, 10 min), the soluble cell-free extract was
heat-precipitated for 30 min at 85° C. Samples were centrifuged
(17,000 × g, 10 min), and the supernatant was applied
to a ResourceQ column (Amersham Pharmacia Biotech, Uppsala, Sweden) and
eluted with 0 to 1.0 M NaCl gradient in 100 mM
Bicine/10 mM MgCl2 buffer using AKTA explorer 10S (Amersham Pharmacia Biotech). Purified Rubisco was applied to
Superdex 200HR 10/30 column in 50 mM sodium phosphate,
0.15 M NaCl buffer (pH 7.0).
Measurement of Rubisco Activity--
3.68 µg of enzyme in 40 µl of 100 mM Bicine/KOH (pH 8.3), 10 mM
MgCl2 buffer, and 10 µl of 15 mM ribulose
1,5-bisphosphate (Sigma) in 100 mM Bicine/KOH (pH 8.3)
buffer were incubated under a 100% CO2 atmosphere in the
experiments described in Fig. 4. Enzyme and substrate were incubated in
air in order to determine the
values in Table I. All buffers were
prepared at the temperatures at which activity measurements were
conducted. Enzyme and substrate were incubated at the desired
temperature for 4 and 1 min, respectively, in closed vials. After
incubation, the two solutions were mixed, and the enzymatic reaction
was carried out at the same temperature for 5, 10, and 15 min. The
products of carboxylase and oxygenase reaction (3-phosphoglycerate or
3-phosphoglycerate and phosphoglycolate for the carboxylase and
oxygenase reaction, respectively) were detected and quantified using
ASAHI PAK GS-220HQ column (Showa Denko K.K., Tokyo, Japan) by UV
absorbance at 210 nm following high performance liquid chromatography
(Shimadzu, Kyoto, Japan). Standards of 3-phosphoglycerate and
phosphoglycolate were added after each measurement, and an additional
run was conducted in order to confirm the retention time of each
compound. Thermostability measurements were conducted by incubating the
enzyme in 100 mM Bicine/KOH (pH 8.3), 10 mM
MgCl2 buffer for the desired time prior to saturation with
CO2. Activity without heat treatment and residual activity
after heat treatment to examine thermostability were measured at
60° C.
Northern Blot Analysis--
A probe corresponding to the
complete open reading frame of Pk-rbc1 was constructed using
the DIG DNA Labeling and Detection Kit (Boehringer Mannheim). Hybond-N+
membranes (Amersham Pharmacia Biotech) were used for blotting. 25 µg
of total RNA was applied to agarose gel electrophoresis.
Western Blot Analysis--
Polyclonal rabbit antibodies were
raised against purified recombinant Pk-Rubisco. Antisera
diluted
500-fold were used to detect Pk-Rubisco in
cell-free extracts of P. kodakaraensis KOD1. Polyvinylidene
difluoride membranes from ATTO (Tokyo, Japan) were used for blotting.
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RESULTS |
Sequence Analysis and Comparison of Structures of Pk-Rubisco with
Other Rubiscos--
Through sequence analysis of the genome of
P. kodakaraensis KOD1, we found an open reading frame of
1332 base pairs (Pk-rbc1), which showed similarity with
previously characterized genes encoding Rubisco. The deduced amino acid
sequence consisted of 444 residues, corresponding in size to the large
subunit of previously characterized Rubiscos (Fig.
1). We compared the amino acid sequence
of Pk-Rubisco with other previously reported enzymes. Type I
and type II enzymes showed high similarity when sequences were compared
among Rubiscos of the same type. Rubisco from spinach (Spinacia
oleracea) (17) showed 83.7 and 71.6% similarity with the type I
enzymes from T. denitrificans (4) and R. sphaeroides (18), respectively. The type II enzyme from R. rubrum (19) displayed 88.2 and 82.9% similarity with the type II
enzymes from R. sphaeroides (20) and T. denitrificans (4), respectively. However, spinach Rubisco showed
only 43.3% similarity with the type II enzyme from R. sphaeroides, indicating that type I and type II enzymes are
distinct in primary structure. In Fig. 2,
we compared Pk-Rubisco with Rubisco from spinach as a
typical type I enzyme, and the type II Rubisco from the
-purple
bacterium R. rubrum. As P. kodakaraensis KOD1 is a hyperthermophilic archaeon, we also included three putative Rubisco
sequences from archaea. One from M. jannaschii showed 58.8%
similarity to the large subunit of Synechococcus PCC6301 Rubisco (8), and two from the chemoautotrophic sulfate-reducing archaeon A. fulgidus (rbcL-1 and
rbcL-2) showed 41 and 45% identity with the M. jannaschii Rubisco (9). From sequence comparison, active site
residues, namely Lys201, Asp203,
His294, and Lys334 of the enzyme from spinach,
were conserved among all protein sequences (Fig. 2). As for
Pk-Rubisco, 51.4% similarity was observed with spinach
Rubisco and 47.3% similarity with the type II enzyme from R. rubrum, suggesting that Pk-Rubisco was not a member of either type. Interestingly, Pk-Rubisco showed 81.9%
similarity with the putative Rubisco RBCL-2 of A. fulgidus.
Pk-Rubisco showed lower similarities, 61.3 and 50.1%, with
M. jannaschii Rubisco and RBCL-1, respectively. Amino acid
residues showing similarity among archaeal Rubiscos were also well
conserved in the type I and type II Rubisco enzymes. There were no
notable regions where high similarity was found only between archaeal
enzymes and type I or type II enzymes.

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Fig. 1.
Nucleotide sequence and deduced amino acid
sequence of Pk-rbc1. The total nucleotide
sequence was confirmed by sequence analysis on both DNA strands.
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Fig. 2.
Amino acid sequence alignment of
Pk-Rubisco with various Rubiscos from other
sources. Sequences compared were type I Rubisco from spinach
(Sp), type II enzyme from R. rubrum
(Rr), three putative Rubisco sequences from archaea, RBCL-1
and RBCL-2 from A. fulgidus (Af1 and
Af2) and M. jannaschii (Mj).
Residues with similarity among archaeal enzymes are indicated with
bars. Conserved residues among archaeal and type I or type
II enzymes are indicated with an asterisk. Active site
residues referred to in the text are indicated with #. Gaps are
indicated with a minus.
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Expression and Purification--
As the structure of
Pk-Rubisco was distinct from type I and type II Rubiscos, we
expressed Pk-rbc1 in E. coli in order to examine
its catalytic activity. High levels of a protein with a molecular mass
of approximately 48 kDa was observed on SDS gels (Fig.
3, lane 2). Following heat
precipitation of the cell-free extract, we were able to purify the
protein by ion exchange and gel filtration chromatography (Fig. 3,
lane 5). The N-terminal sequence of the protein was
Val-Glu-Lys-Phe-Asp-Thr-Ile-Tyr-Asp-Tyr-Tyr, which was identical to the
predicted sequence beginning with the second amino acid residue deduced
from the open reading frame. We also investigated the subunit
composition of the enzyme by gel filtration chromatography. The native
enzyme had a molecular mass of 450 kDa, indicating that
Pk-Rubisco was an L8 type homo-octamer (data not
shown).

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Fig. 3.
Gene expression of Pk-rbc1
in E. coli and purification of
Pk-Rubisco. pET21a(+) inserted with
Pk-rbc1 was used to transform E. coli BL21(DE3),
and gene expression was induced with
isopropyl-D-thiogalactopyranoside. Cell-free extracts (10 µg) were harvested 0 h (lane 1) and 4 h
(lane 2) after induction, and the supernatant (2.5 µg)
after heat precipitation (lane 3), eluate (2.5 µg) after
anion exchange column ResourceQ (lane 4), and purified
Pk-Rubisco (5.0 µg) after gel filtration chromatography
(lane 5) were applied to SDS-polyacrylamide gel
electrophoresis. The arrowhead indicates the protein product
Pk-Rubisco.
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Characterization of Pk-Rubisco--
We next investigated the
enzymatic activity of the purified Pk-Rubisco. Measurements
of carboxylase activity were conducted at various temperatures. We
could clearly detect significant carboxylase activity from 40 to
100° C, and the optimum temperature was extremely high, at 90° C
(Fig. 4A). The optimum pH of
the enzyme was 8.3 (data not shown). The enzyme also showed high
thermostability, with a half-life of 2.5 h at 100° C and
15 h at 80° C (Fig. 4B). The specific activity of
Pk-Rubisco at 90° C was 19.8 × 103 nmol
of CO2 fixed per min/mg (Fig. 4A). This specific
activity level is higher than any previously characterized Rubisco.
Another important parameter in the case of Rubisco is the
value,
which is the ratio of carboxylase activity to oxygenase activity. The
value of Pk-Rubisco was 70 at 50° C, 140 at 60° C,
250 at 70° C, 290 at 80° C, and 310 at 90° C (Table
I). The increase in
value of
Pk-Rubisco at elevated temperatures is totally distinct from
previously characterized Rubiscos, whose
values decrease with
increase in temperature (21).

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Fig. 4.
Characterization of
Pk-Rubisco. A, temperature profile of
carboxylase activity of Pk-Rubisco. Measurements were
conducted at pH 8.3. B, thermostability of
Pk-Rubisco at 80° C (closed boxes) and
100° C (closed circles). Carboxylase activity prior to
heat treatment was defined as 100% and relative residual activities
(%) after heat treatments were calculated. All activity measurements
were conducted under saturating CO2 conditions, as
described under "Materials and Methods."
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Detection of Pk-Rubisco in P. kodakaraensis KOD1--
In order to
determine whether Pk-rbc1 was actually expressed in the host
cells, we conducted Northern blot analysis using a DNA probe
corresponding to the complete open reading frame of Pk-rbc1.
An mRNA with a length of 1,400 bases specifically hybridized with
the Pk-Rubisco probe (Fig.
5A), confirming that
Pk-rbc1 is transcribed in the native host. The length of the
mRNA strongly indicates that Pk-rbc1 is transcribed as a
single gene and not a member of a multi-gene operon. In order to
conduct Western blot analysis, polyclonal antibodies were raised
against the recombinant Pk-Rubisco. A specific band with a
molecular mass identical with the recombinant protein and corresponding
well with the size of Pk-Rubisco deduced from its amino acid
sequence (49,710 Da) was observed (Fig. 5B). The above
results prove that Pk-rbc1 is transcribed and translated and
that the protein product, Pk-Rubisco, is present in the
native host, P. kodakaraensis KOD1.

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Fig. 5.
Northern blot analysis for mRNA of
Pk-Rubisco (A) and Western blot
analysis of Pk-Rubisco (B).
A, 25 µg of total RNA was used for agarose gel
electrophoresis. B, recombinant Pk-Rubisco
(lane 1, 60 ng) and cell-free extract of P. kodakaraensis KOD1 (lane 2, 60 µg) were applied to
SDS-polyacrylamide gel electrophoresis followed by blotting to the
polyvinylidene difluoride membrane. Arrows in A
and B indicate the detected bands.
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DISCUSSION |
We have identified and characterized a Rubisco gene from the
hyperthermophilic archaeon P. kodakaraensis KOD1. By
expression of the gene and activity measurements of the protein
product, we have demonstrated that the gene actually encodes an active Rubisco enzyme. Although putative Rubisco genes from the archaea M. jannaschii and A. fulgidus have also been
recently reported through genome analysis (8, 9), identification of the
translation products has not been reported as of yet. Therefore, the
question remains whether they actually encode active Rubiscos.
Pk-Rubisco proved to be an extremely thermostable enzyme,
and its specific activity at 90° C was the highest among all
previously characterized Rubiscos. This may primarily be due to the
high temperature where activity tests were carried out. Kinetic studies indicated that Pk-Rubisco had a
value of 310, highly
specific toward the carboxylase reaction. Type I Rubiscos from plants
have a
value of approximately 90, while those from
Chromatium or Synechococcus share a value of
approximately 45 (22). Recently, a Rubisco from thermophilic red algae
was reported to possess a high
value of 238 at 25° C, which
decreased when temperature was elevated (21). In contrast, the
value of Pk-Rubisco increased as temperatures were elevated
(Table I). Future studies on the kinetics and three-dimensional
structure of the protein are necessary to understand the structural
characteristics of Pk-Rubisco which lead to the extremely
high activity and
value. This should also identify the presence of
ion pair networks involved in the high thermostability of
Pk-Rubisco.
We have not been able to identify an ORF with similarity to the small
subunit of Rubisco. Results of Northern blot analysis indicated that
Pk-rbc1 was not a member of a multi-gene operon and the most
nearby ORFs encoded histone and enzymes involved in tryptophan
biosynthesis (data not shown). At present, considering that
Pk-Rubisco displayed high carboxylase activity as a
homo-octamer, we expect Pk-Rubisco be composed of only large
subunits. In support, ORFs showing similarity with the Rubisco small
subunit could not be found in the whole genome of M. jannaschii and A. fulgidus.
Western blot analysis has proven that Pk-Rubisco is present
in P. kodakaraensis KOD1. Therefore, identification of the
physiological role of Pk-Rubisco, especially its
contribution to carbon dioxide fixation, will be an attractive target
in future research. P. kodakaraensis KOD1 was isolated from
a solfatara deep under sea level, and at present we have no evidence
that the strain can grow chemolithotrophically. We found high
structural similarity of Pk-Rubisco with the enzyme from
A. fulgidus, and A. fulgidus can grow
lithoautotrophically on hydrogen, thiosulfate, and carbon dioxide.
Genome analysis of A. fulgidus has revealed the presence of
putative ribose-5-phosphate isomerase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase genes of the Calvin cycle (9). The corresponding genes have
also been found in M. jannaschii (8). However, a putative
ribulose-5-phosphate kinase, whose product should be the substrate of
Rubisco, has not been identified in either strain. Further metabolic
studies of P. kodakaraensis KOD1 are necessary to identify
the pathway to which Pk-Rubisco mainly contributes.
The primary structure of Pk-Rubisco was clearly distinct
from those of type I and type II enzymes. The
value was also
significantly higher than those of type I and type II Rubiscos. As we
have proven that the protein actually harbors Rubisco activity, we
regard Pk-Rubisco as a structurally novel Rubisco. The
putative Rubisco RBCL-2 of the archaeon A. fulgidus was the
most closely related enzyme, showing 81.9% similarity. Furthermore,
phylogenetic analysis of various Rubiscos indicated that the archaeal
enzymes, although widely diverse, followed a distinct pathway of
evolution (data not shown). These results suggest that archaeal
Rubiscos, if others are expressed and show activity as in the case of
Pk-Rubisco, may constitute a structurally distinct type of Rubisco.