Department of Microbiology and Plant
Biotechnology Center, The Ohio State University, Columbus, Ohio
43210-1292
Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of
the Calvin-Benson-Bassham cycle may be divided into two broad
phylogenetic groups, referred to as red-like and green-like, based on
deduced large subunit amino acid sequences. Unlike the form I enzyme
from the closely related organism Rhodobacter sphaeroides, the form I RubisCO from R. capsulatus is a member of the
green-like group and closely resembles the enzyme from certain
chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic
properties of this type of RubisCO have not been well studied in a
system that offers facile genetic manipulation, we purified the
R. capsulatus form I enzyme and determined its basic
kinetic properties. The enzyme exhibited an extremely low substrate
specificity factor, which is congruent with its previously determined
sequence similarity to form I enzymes from chemoautotrophs and
cyanobacteria. The enzymological results reported here are thus
strongly supportive of the previously suggested horizontal gene
transfer that most likely occurred between a green-like
RubisCO-containing bacterium and a predecessor to R. capsulatus. Expression results from hybrid and chimeric enzyme
plasmid constructs, made with large and small subunit genes from
R. capsulatus and R. sphaeroides, also
supported the unrelatedness of these two enzymes and were consistent
with the recently proposed phylogenetic placement of R. capsulatus form I RubisCO. The R. capsulatus form I
enzyme was found to be subject to a time-dependent fallover in activity
and possessed a high affinity for CO2, unlike the closely
similar cyanobacterial RubisCO, which does not exhibit fallover and
possesses an extremely low affinity for CO2. These latter
results suggest definite approaches to elucidate the molecular basis
for fallover and CO2 affinity.
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INTRODUCTION |
It is well established that the
synthesis of both form I and form II ribulose 1,5-bisphosphate (RuBP)
carboxylase/oxygenase (RubisCO) from the nonsulfur purple bacterium
Rhodobacter capsulatus is highly dependent on the
environmental growth conditions (8, 33). Yet, aspects of the
molecular biology of autotrophic carbon dioxide metabolism and RubisCO
synthesis in this organism have only recently been stressed
(26-28), and there is little information available on the
enzymology of RubisCO in this versatile organism. This neglect has
largely been due to the fact that the regulation and biochemistry of
CO2 assimilation are highly studied in the closely related
organism Rhodobacter sphaeroides (6, 16, 31, 36).
Early immunological studies, however, indicated that there were
substantial and unexpected differences between the form I enzymes of
R. capsulatus and R. sphaeroides (8),
which was later substantiated by DNA hybridization results
(34) and additional immunological studies (26).
The distinctness of the form I RubisCO of R. capsulatus was
recently confirmed during a recent study of the organization and
regulation of the CO2 fixation genes of this organism
(28). During this investigation, it was noted that deduced
amino acid sequences of the large and small subunits of form I RubisCO
from R. capsulatus and R. sphaeroides were
surprisingly different, despite the close phylogenetic relatedness of
the two organisms based on other molecular indicators (27). In fact, the sequence results placed the R. capsulatus form
I RubisCO among members of what has been termed the green-type RubisCO group (4, 38), which is comprised of 
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chemoautotrophic proteobacterial and cyanobacterial enzymes
(27). It is apparent that all genes of the
cbbI regulon of R. capsulatus are
derived from chemoautotrophic bacteria via some form of horizontal gene transfer (27), whereas genes of the
cbbII regulon of this organism closely resemble
those from its close relative R. sphaeroides (21,
28). Because of the potential uniqueness of form I RubisCO of
R. capsulatus and the availability of recombinant clones
(26, 28), it was suggested that detailed study of this
enzyme might yield useful insights concerning important molecular
properties of a class of RubisCO that has not been highly studied to
this point (36). Such studies are important because the
determinants responsible for discrimination between either
CO2 or O2 as gaseous substrate are largely
unknown. The capacity to preferentially use either CO2 or
O2 is a property that has great physiological relevance,
and yet it can vary significantly depending on the evolutional form of
the enzyme (36). Thus, the potential to use the great
genetic capacity of R. capsulatus to learn more of the
molecular basis for CO2/O2 specificity for an
unusual type RubisCO about which little is known was an important
consideration in initiating such studies. Finally, despite the relative
unrelatedness of R. capsulatus and R. sphaeroides
form I RubisCO amino acid sequences (58% identity), the large subunit
genes from each organism (of similar GC content) are colinear over the
majority of the coding region. These sequences would thus be quite
amenable for the construction of molecular chimeras or large
subunit-small subunit hybrids without disrupting the reading frame.
Such studies would be important if kinetic and other properties of the
two form I enzymes, especially the CO2/O2
substrate specificity, proved to be different. For these reasons, an
investigation of the molecular properties and enzymology of form I
RubisCO from R. capsulatus was initiated. Because the
enzymological properties of this enzyme were found to be unique,
preliminary efforts were also begun to produce hybrid large
subunit-small subunit R. capsulatus-R. sphaeroides form I
enzymes and molecular chimeras, with the eventual goal of pinpointing
molecular determinants responsible for key properties.
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MATERIALS AND METHODS |
Growth of R. capsulatus and E. coli
strains.
R. capsulatus wild-type strain SB1003 and form II
RubisCO-deficient mutant strain SBII
(28) were
grown photoautotrophically in Ormerod's minimal medium (25)
supplemented with kanamycin (5 µg/ml) as previously described (26). Cells were grown to late log phase (optical density at 600 nm of >1.0) in 0.5-liter batch cultures, harvested by
centrifugation, and washed with TEDM (25 mM Tris-Cl [pH 8.0], 1 mM
EDTA, 10 mM MgCl2, 2 mM dithiothreitol [DTT]).
All plasmid propagation and protein expression studies were performed
in Escherichia coli JM109 (39). E. coli(pKF1BP), conferring lac promoter-controlled
expression of the R. capsulatus form I RubisCO
(26), was grown in 0.5-liter Luria-Bertani medium batch cultures supplemented with kanamycin (25 µg/ml). Expression of recombinant RubisCO was induced at an optical density at 600 nm of 0.5 by the addition of isopropyl--D-thiogalactopyranoside to
a final concentration of 0.5 mM. Cells were harvested after a 20-h
induction period by centrifugation and washed as stated above.
Enzyme purification.
Crude lysates were prepared from washed
cell pellets resuspended (1 ml/g [wet weight]) in TEDM adjusted to 1 mM phenylmethylsulfonyl fluoride (added just before cell breakage).
Cell suspensions were passed through a French pressure cell (SLM
Instruments, Inc., Urbana, Ill.) at 16,000 lb/in2 three
times, and cell debris was removed by centrifugation at 12,000 × g to yield a clear cell lysate. Contaminating
chromosomal DNA in the clear cell lysate was hydrolyzed by incubating
the extract with DNase I (100 µg/ml) for 30 min at room temperature. All subsequent purification steps were carried out at 4°C unless otherwise indicated. A soluble protein extract was then obtained after
centrifugation at 100,000 × g for 1 h; the
supernatant was retained and RubisCO was partially purified from this
fraction by Green A (Amicon, Beverly, Mass.) dye affinity
chromatography using a 2.5- by 4-cm column equilibrated in TEMMB (25 mM
Tris-HCl [pH 7.2], 1 mM EDTA, 5 mM -mercaptoethanol, 10 mM
MgCl2, 50 mM NaHCO3). The Green A column was
washed with equilibration buffer until the A280
of the eluent returned to baseline (~250 ml); RubisCO was then eluted
with a 300-ml linear salt gradient (0 to 1 M KCl) in TEMMB. Peak
fractions were pooled and concentrated in an Amicon Centriplus 30 concentrator. RubisCO was purified to homogeneity by loading the
concentrated Green A peak onto a Pharmacia Mono Q HR 5/5 column
equilibrated with 10 mM KCl in TEM (TEDM minus DTT) and subjected to a
5-ml 10 to 500 mM KCl gradient. RubisCO-containing fractions were
desalted using a Bio-Rad (Richmond, Calif.) DG10 column equilibrated
with 20% glycerol in TEM (at room temperature), quickly frozen with
liquid nitrogen, and stored at 
70°C.
Purity of enzyme preparations was assessed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 15% resolving gels (20). Western immunoblots were prepared by
transferring SDS-PAGE-resolved proteins onto Immobilon-P polyvinylidene
difluoride membranes (Millipore, Bedford, Mass.), using a Bio-Rad
Trans-Blot SD semidry electrophoretic transfer cell with either the
transfer buffer of Towbin (37) (25 mM Tris, 192 mM glycine,
20% methanol [pH 8.3]) or CAPS
[3-(cyclohexylamino)-1-propanesulfonic acid] transfer buffer (10 mM
CAPS, 10% methanol [pH 11.0]) for N-terminal sequencing. Blots were
blocked and washed with phosphate-buffered saline (0.8% NaCl, 0.02%
KCl, 0.115% NaH2PO4 · 7H2,
0.02% KH2PO4)-0.05% Tween 20. Primary
antibodies directed toward R. sphaeroides and Synechococcus strain PCC 6301 RubisCO and commercially
prepared (Bio-Rad) secondary antibody were all used at 1:3,000
dilutions in PBS phosphate-buffered saline-Tween 20. After exposure to
primary and secondary antibodies, blots were briefly equilibrated in
0.1 M Tris-acetate (pH 9.5) before incubation in Attophos (2 min). Signal and documentation of chemifluorescent Western blots were achieved with a Molecular Dynamics (Sunnyvale, Calif.) model 864 Storm
PhosphorImager. The N-terminal sequence of CbbL was determined with an
Applied Biosystems (Foster City, Calif.) 475A protein sequencer at The
Ohio State Biopolymer facility.
RubisCO was assayed as previously described (7) and
according to conditions specified in the figure legends and tables.
Subcloning RubisCO large and small subunit genes.
To
simplify future R. capsulatus-R. sphaeroides RubisCO chimera
constructions involving EcoRI, all genes were subcloned into a derivative of the pK19 expression vector (30), pK19
E1,
which lacks an EcoRI site (this study). The cbbS
gene of R. sphaeroides (cbbSs) was
subcloned as an NruI fragment from pRSF1A, which contains both the large and small subunit genes from R. sphaeroides,
into the SmaI site of pK19E1, generating plasmid pRsS-17
(Fig. 1). Proper orientation of the
insert with respect to the lac promoter was confirmed by DNA
sequencing with the universal forward primer. The R. sphaeroides RubisCO large subunit gene
(cbbLs) was PCR amplified by using pRSF1A as
a template and custom forward (5'-GGAAGCTTA TGCTTCGAAGATCACCG-3') and reverse
(5'-GGTCTAGAAGCAGCCTTGGGTGA TGC-3') primers
(Operon Technologies, Inc., Alameda, Calif.) that introduced unique
5' HindIII and 3' XbaI sites (underlined),
respectively. Optimal synthesis of the desired 1.6-kb product was
obtained under the following reaction conditions: 1× U.S. Biochemical
(Cleveland, Ohio) Taq polymerase buffer, 200 µM
deoxynucleoside triphosphates, 1.7 pmol of template (5.22 kb), 0.4 pmol
of primers, 1.0 mM MgCl2, and 5 U of Taq
polymerase (U.S. Biochemical). The PCR program was 3 min at 95°C for
the denaturation step; 30 cycles of 1 min at 95°C, 1 min at 60°C,
and 3 min at 72°C; and 7 min at 72°C for the extension step. The
resultant 1.6-kb PCR product was subcloned into pUC19 (39)
as an HindIII/XbaI fragment, generating
plasmid pRsL-1 (Fig. 1).
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FIG. 1.
Diagrammatic representation of hybrid and wild-type
RubisCO holoenzyme expression plasmids. The plasmids were named such
that the origin of each subunit gene is indicated by the subscripted
first letter of the species from which it originated. The bold arrow
indicates the location of the lac promoter with respect to
the cbb genes, which are all in the proper transcriptional
orientation for controlled expression with this promoter. Open and
shaded rectangles represent coding regions originating from R. sphaeroides and R. capsulatus, respectively.
Abbreviations for restriction sites: H, HindIII; X,
XbaI; B, BamHI; and K, KpnI.
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The form I RubisCO genes of R. capsulatus
(cbbLc and cbbSc) were
also amplified individually (using PCR) in order to incorporate unique
restriction sites for directional cloning, using primers pcbbLc forward
(5'-AAGCTTAAGTCCCGCAAGGCC-3') and reverse
(5'-TCTAGATGTCCTTTCGGGCCG-3') and
pcbbSc forward
(5'-GGGGATCCTGCAGCACCGCTGAG-3') and reverse (5'-GGGGTACCGATGGGGAAAGGGGC-3'), introducing
BamHI and KpnI sites, (underlined), respectively.
The PCRs for these two genes were optimized for Mg2+
concentration (cbbLc, 1.0 mM;
cbbSc, 1.5 mM), using otherwise identical
reaction conditions as specified above. PCR product size and direct
restriction endonuclease digest banding patterns were consistent with
expectations for the desired products. The cbbSc
PCR product was purified with a Wizard PCR Prep kit (Promega Corp.,
Madison, Wis.) and subcloned with a pCR-Script Amp SK(+) cloning kit as
recommended by the manufacturer (Stratagene, La Jolla, Calif.).
Briefly, the blunt-ended PCR fragment was ligated into pCR-Script Amp
SK(+), generating pRcS-12. The cbbSc gene was
then subcloned as a BamHI/KpnI (sites introduced
by PCR) fragment into pK19E1, forming pRcS-4 (Fig. 1). R. capsulatus cbbL was never successfully subcloned after numerous
attempts using all of the methods described above.
All PCR primers and subcloning steps described here were designed such
that none of the constructs resulted in translational fusions with the
lacZ peptide.
Construction of wild-type, hybrid, and chimera RubisCO expression
plasmids.
The restriction site incorporation and cloning steps
described above facilitated the simple construction of wild-type (used as positive controls in expression experiments) and hybrid holoenzyme expression plasmids (diagrammatically represented in Fig. 1). To
reconstruct an R. sphaeroides cbbL-cbbS expression plasmid (pLsSs), the large subunit gene
(cbbL) was directionally subcloned as a
HindIII/XbaI fragment from pRsL-1 into
pRsS-17 (Fig. 1). Similarly, to express a hybrid between R. sphaeroides large and R. capsulatus small subunits,
cbbLs was subcloned upstream of R. capsulatus cbbS (cbbSc) in pRcS-4,
producing plasmid pLsSc (Fig. 1). Restriction
digests and DNA sequencing of the 5' and 3' ends of both plasmid
inserts (pLsSs and
pLsSc) with universal and reverse primers
confirmed that the desired clones were made (data not shown). In
addition, a large subunit chimera construct was made between R. sphaeroides and R. capsulatus, using a colinear XhoI restriction site in both genes (Fig. 1). Plasmid
pQ, containing the R. capsulatus cbbL and
cbbS genes, was digested with
HindIII/XhoI and ligated to the ~1.0-kb
HindIII/XhoI fragment (result of a partial
digest with XhoI) of pRsL-1 (containing the R. sphaeroides cbbL gene), producing plasmid ps/cL-Xho1, which contains a chimeric cbbL gene and a wild-type R. capsulatus cbbS gene (Fig. 1). Restriction digests and DNA
sequencing of the insert's 5' and 3' ends confirmed that the desired
clone was obtained (data not shown). In addition, an approximately 650 bp EcoRI fragment of ps/cL-Xho1 (which would not exist in
the parent plasmid, pQ) that encompasses the splice site
of the chimeric cbbLs/c was subcloned and
sequenced to confirm that the desired chimera was constructed.
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RESULTS AND DISCUSSION |
RubisCO gene expression and enzyme purification.
The highest
levels of form I RubisCO (~15% of soluble protein) were obtained
from R. capsulatus SBII, a strain containing
an inactivated form II (cbbM) RubisCO gene. This was
particularly evident when strain SBII was grown under
photolithoautotrophic conditions to late log phase (data not shown).
Since previous studies (28) had indicated that the absence
of form II RubisCO leads to a compensatory increase in form I RubisCO
synthesis, these results were not surprising. However, it was further
observed that 90 to 95% of the RubisCO activity was found in the
particulate fraction when cell extracts were subjected to
centrifugation at 100,000 × g. This phenomenon was
observed when the form I RubisCO was prepared both from R. capsulatus (SB1003 and SBII) and from E. coli FIPB; thus, the potential formation of carboxysomes in
R. capsulatus to account for these results was ruled
unlikely. This is in agreement with Southern hybridization data that
showed no evidence for a ccmK homologue (a gene product
associated with carboxysome structures) when R. capsulatus
chromosomal DNA was probed at very low stringency (25a).
Western analysis (results not shown) and staining of SDS-polyacrylamide
gels containing extracts from photolithoautotrophically grown R. capsulatus SB1003 (a growth condition where both forms of RubisCO
are expressed), before and after centrifugation at 100,000 × g, established that the sedimentation phenomenon was specific
for the form I enzyme. This is indicated by a decrease in the relative
amount of form I protein in the 100,000 × g fraction
(Fig. 2). Further experimentation demonstrated that this enzyme had an unusually high affinity for DNA,
causing it to cosediment with chromosomal DNA during high-speed centrifugation. The majority of the enzyme activity in cell extracts could be recovered in the 100,000 × g supernatant if
the extract was pretreated with DNase I to hydrolyze contaminating
chromosomal DNA. Other pretreatments, such as incubation in the
presence of 1% Nonidet P-40, 0.5 M NaCl, and 10 mM EDTA, did not
prevent sedimentation (data not shown). Application of the
SBII soluble protein extract to a Green A dye affinity
column, washing with TEMMB (pH 7.2), and elution with a KCl gradient
(Table 1) resulted in a 1.6-fold
purification. Mono Q anion-exchange chromatography of the Green A peak
material yielded an enzyme preparation that was judged greater than
95% pure by SDS-PAGE (results not shown) and exhibited a specific
activity of >3.0 U/mg under optimal assay conditions. N-terminal
sequencing, after elution of SDS-PAGE-purified large subunits from
gels, corresponded well with the deduced amino acid sequence
(27) except for a missing N-terminal methionine residue as
seen with other sequenced RubisCO large subunits (19). The
purified enzyme was desalted, adjusted to 20% glycerol, quickly frozen
in liquid N2, and stored without loss of activity at
70°C for months.
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FIG. 2.
SDS-PAGE of R. capsulatus SB1003 cell
extracts and purified form I RubisCO. Proteins were electrophoresed in
a 15% denaturing polyacrylamide resolving minigel; 15 µg of cell
extract (lanes 1 and 2) and 1 µg of purified holoenzyme (lane 3) were
loaded. Small subunits were electrophoresed off this gel to enhance the
resolution between form I and form II large subunits (LSU; bands
indicated on the left). Lanes 1 and 2 were loaded with
10,000 × g and 100,000 × g
supernatant fractions, respectively.
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Enzymatic characterization of R. capsulatus form
I RubisCO.
The activity of purified R. capsulatus
form I RubisCO was particularly sensitive to low protein concentrations
(<5 µg/ml), as only about 30% of its optimal activity was obtained.
Activity increased by 50% with the addition of DTT (5 mM) and
increased by 40 to 370% (depending on the amount of RubisCO assayed)
when both DTT and bovine serum albumin (100 µg/ml) were included in reaction mixtures (results not shown). When the enzyme was carbamylated to convert the enzyme to an active ternary complex (24),
i.e., by preincubation in the presence of Mg2+ and
CO2, the highest activities were obtained at 30°C in the presence of 5 mM DTT (activity increased by 152% over the untreated enzyme). The presence of DTT improved activity at all temperatures but
was maximally effective at 30°C. Preincubation temperatures of 37 and
48°C progressively inhibited enzyme activity (activity decreased by
92% in the absence of DTT).
With the exception of the Synechococcus strain PCC 6301 enzyme and other cyanobacterial enzymes, all other form I RubisCO enzymes characterized to date are inhibited by the presence of RuBP in
preincubations with uncarbamylated (unactivated) enzyme (36). This phenomenon has been clearly shown to be
attributed to the ability of RuBP to lock the unactivated enzyme to a
state that prevents the necessary carbamylation (activation) of the enzyme when CO2 and Mg2+ are subsequently added
(29). Cyanobacterial form I RubisCO and Thiobacillus
denitrificans form I RubisCO, which are both closely related at
the primary sequence level to the R. capsulatus form I
enzyme, clearly exhibit linear and noninhibited reaction time courses
when RuBP is added to preincubation mixtures (11, 23). The
rate of the R. capsulatus form I RubisCO, however, was
substantially inhibited by the presence of substrate RuBP in similar
preincubation experiments (Fig. 3).
Moreover, the clear decrease in the reaction rate of
CO2/Mg2+-preincubated R. capsulatus
enzyme after about 2 to 3 min is diagnostic of fallover due to the
formation of high-affinity protonated RuBP intermediates during
catalysis that inhibit the reaction. Fallover is not observed with the
cyanobacterial or T. denitrificans enzymes (11,
23). Thus, despite the primary sequence similarities of the
cyanobacterial/thiobacillus and R. capsulatus form I
RubisCOs, these results represent clear differences between these
enzymes.
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FIG. 3.
Preincubation and effect of RuBP on the activation of
R. capsulatus form I RubisCO. Enzyme was desalted with a
Bio-Rad DG10 column and preincubated either in the presence of standard
concentrations of magnesium and bicarbonate or in the presence of RuBP
for 5 min before initiation of the reaction with the components needed
to complete the assay mixture (23). At the indicated times,
aliquots of the assay mixture were acidified and then counted by liquid
scintillation spectroscopy; 0.5 µg of purified enzyme was used in the
reaction.
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Further kinetic characterization of the R. capsulatus form I
RubisCO indicated that the KRuBP was 8 µM,
using standard Michaelis-Menten procedures (Table
2). Examination of other kinetic
properties, however, yielded unexpected results. The
CO2/O2 substrate specificity factor (
or
) of the R. capsulatus form I enzyme was found to be 26, as determined by the dual-label assay method of Jordan and Ogren
(15). This value is much lower than that obtained for the
unrelated form I enzyme of R. sphaeroides and somewhat lower
than that of the sequence-similar Synechococcus strain PCC 6301 enzyme (Table 2) and form I enzyme of T. denitrificans
(11). A limited number of L8S8 (form
I) enzymes have been studied with specificity factors below 30. Among
all sequences in the database, the deduced amino acid sequence of
Pseudomonas hydrogenothermophila (40) exhibited
the highest degree of similarity (87% identity) to the R. capsulatus catalytic subunit. Not surprisingly, the values
recently reported for two Hydrogenovibrio marinus form I
enzymes, which are also highly similar (78 and 76% identity, respectively), were also rather low, at 27 and 33, respectively (13).
It is well established that several cyanobacteria (including
Synechococcus) and some aerobic chemoautotrophic bacteria
form structures known as carboxysomes, which are cellular inclusions containing aggregates composed largely of paracrystalline RubisCO. No
such structures have been described for R. capsulatus. The proposed function of the carboxysomes in organisms that bear them is to
facilitate the transport and concentration of carbon dioxide to RubisCO
active sites (17). Interestingly, the
KCO2 for the R. capsulatus form I enzyme was determined to be 29 µM (Table 1).
Despite the fact that the cyanobacterial and R. capsulatus form I RubisCOs are so homologous (72 and 37% identity for large and
small subunits, respectively), the cyanobacterial enzyme has a much
lower affinity for this gaseous substrate
(KCO2 = 174 µM [Table 2]),
as does the enzyme of T. denitrificans (11). The low KCO2 of the R. capsulatus enzyme may explain why an organism like R. capsulatus, containing an inactivated form II RubisCO gene
(28), is able to grow so efficiently under chemoautotrophic conditions in the presence of oxygen even though it possesses a low
CO2/O2 substrate specificity form I RubisCO and
has no apparent carbon concentrating mechanism. It would appear that
selection pressures favored the evolution of this type of enzyme. Most
importantly, the availability of the R. capsulatus form I
enzyme should stimulate future DNA shuffling experiments (3)
with sequence-related form I enzymes that possess different properties,
such as cyanobacterial (Synechococcus) RubisCO, so that the
molecular basis for varied CO2 affinity and fallover might
be ascertained.
Expression of wild-type, hybrid, and chimeric RubisCOs in E. coli.
Initial steps were made to begin uncovering relevant
structural differences between the form I RubisCOs from R. capsulatus and R. sphaeroides. Although the primary
sequences are somewhat different (27), large subunits and
small subunits still show 58 and 34% identity at the amino acid level.
Few hybrid and chimeric RubisCO mutants have been expressed in vivo to
study structure-function relationships. However, vastly dissimilar
recombinant small subunits from a eukaryotic nongreen alga could
assemble with cyanobacterial large subunits to form a holoenzyme with a
substrate specificity factor enhanced by 60% (32). Despite
a 20-fold loss in carboxylase activity, this hybrid provided insights
concerning individual subunit involvement in regulation by sugar
phosphate metabolites (32). Not all attempts to make hybrid
RubisCOs have been so fruitful. Lee et al. (22) described a
hybrid consisting of cyanobacterial large subunits and small subunits
from the chemoautrophic bacterium Ralstonia eutropha
(Alcaligenes eutrophus) which exhibited similar kinetic
properties (except a 85% reduction in kcat) to
the natural cyanobacterial holoenzyme. Catalytically active hybrids
have also been successfully made in vitro by combining partially
purified large and small subunits (1, 2, 12, 14), but in
most cases Michaelis constants and other enzymological properties were not investigated. In any case, the previous results cited above suggested that in vivo hybrid and chimera construction experiments with
the requisite genes of R. capsulatus and R. sphaeroides, which are more closely related than the above
algal-cyanobacterial hybrid components, might be quite interesting. In
addition, the sequences from R. capsulatus and R. sphaeroides do present some advantages for such constructions,
including the ability to make facile chimeras without disrupting the
reading frame. Identifying regions of the RubisCO molecule that are
germane to the various properties discussed above may help explain the
disparity in specificity values and other kinetic parameters.
Eventually, it is hoped that these types of studies will suggest the
participation of new residues or domains that have so far gone
unnoticed in studies using current X-ray structural models. Considering
the differences in amino acid sequence (27) and kinetic
properties (Table 2), there was the potential to observe dramatic
effects upon construction of hybrids and chimeras with the genes
encoding large and small subunits of the R. capsulatus and
R. sphaeroides enzymes.
Several constructions were attempted. However, a report indicated that
the cbbQ product, located downstream from
cbbL-cbbS, in P. hydrogenothermophila, is
required for maximum activity and folding of this enzyme (10,
13). Previous work had not established whether recombinant form I
R. capsulatus RubisCO activity and folding in E. coli required coexpression of cbbQ, a gene also present
downstream from cbbL-cbbS of R. capsulatus
(27). Since the previously used RubisCO expression vector
(pKFIBP) contained the cbbQ gene (26), we
constructed an expression plasmid that lacked cbbQ
downstream of cbbL-cbbS (Fig. 1). To determine the necessity
of cbbQ for stable production of recombinant R. capsulatus RubisCO, extracts of induced cells, harboring
plasmids pLSQ-21 (containing the same cbbL-cbbS-cbbQ
insert as pKFIBP) (26) or pQ (encoding only
cbbL-cbbS) (Fig. 1), had virtually identical levels of
RubisCO activity. Specific activities of 18 and 17 mU/mg were obtained
for E. coli(pLSQ-21) and E. coli(pQ), respectively. Thus, these results
indicated that CbbQ was neither required, nor did it enhance RubisCO
activity (or stability) for the recombinant R. capsulatus
form I enzyme.
Sequence differences and other more subtle structural idiosyncrasies
obviously account for variations in the kinetic properties for the
respective R. capsulatus and R. sphaeroides
holoenzymes. Therefore, hybrid and chimeric enzyme expression vectors
were constructed by using the R. sphaeroides and R. capsulatus large (cbbL) and small (cbbS)
subunit genes (Fig. 1). Several unsuccessful attempts were made to
induce the expression of active LsSc hybrid under conditions proven to induce the two parent holoenzymes in cells
with plasmids containing the original cbbL-cbbS encoding chromosomal inserts (Table 3). To ensure
that the cloned genes (cbbLs and
cbbSc) were in fact functional, two measures
were taken. First, the pLsSs expression plasmid
was reconstructed by using the individually cloned genes from pRsL-1
and pRsS-17 (Fig. 1). Extracts from cells containing the resultant
plasmid (pLsSs) efficiently expressed RubisCO
under identical induction conditions (Table 3). SDS-PAGE analysis of
these extracts indicated that cbbLS was highly
overexpressed, and the recombinant proteins comigrated with purified
R. sphaeroides large and small subunits (results not shown).
Second, cell extracts and the urea-solubilized inclusion body fraction
from LsSc hybrid-containing cells were used for in vitro reconstitution assays with E. coli extracts
containing recombinant cyanobacterial large subunits; purified
cyanobacterial small subunits were used as a positive control for small
subunit reconstitution activity (12).
Reconstitution-competent R. capsulatus small subunits were
found in the inclusion body fraction; however, R. sphaeroides large subunits could not be detected by this approach in the LsSc cell extract using purified
cyanobacterial small subunits (Table 3). Taken together, these data
suggest the following. (i) The genes used to make the
LsSc hybrid are functional. When these genes
were expressed together to produce their cognate gene products, active
holoenzyme was assembled. (ii) The R. sphaeroides large and
R. capsulatus small subunits were incapable of forming functional hexadecameric hybrid enzymes, since small subunits were
sequestered in the inclusion body fraction instead of being incorporated into the holoenzyme. The same phenomenon was observed when
a variety of RubisCO small subunit genes were expressed in E. coli in the absence of coexpressed large subunits (12,
35) and when conserved small subunit residues were mutated in the cyanobacterial enzyme from Anabaena strain 7120 (5).
Similar results were obtained from cells synthesizing the large subunit
chimera (ps/cL-Xho1) with R. capsulatus small subunits (Table 3). This large subunit chimera consists of 62% R. sphaeroides sequence from the N terminus, with the remainder
derived from R. capsulatus cbbL (Fig. 1). Crystallography
data for spinach RubisCO shows that virtually all large subunit
residues present at large/small subunit interfaces in the holoenzyme
are contributed from the C-terminal / barrel domain (i.e., the
last ~320 amino acids, which correspond to 67% of the C terminus)
(18). In addition, all large subunit residues involved in
hydrogen bonds between large and small subunits reside in the
C-terminal domain (18). Thus, it was thought that the
chances of this chimera producing a productive holoenzyme were high.
Evidently replacing or substituting 38% percent of the C terminus with
R. capsulatus sequence, however, was not enough to resurrect
an active, stable holoenzyme (Table 3).
The 58% sequence identity (465-amino-acid overlap) between R. capsulatus and R. sphaeroides form I large subunits
suggests that these proteins may have similar tertiary structures.
Indeed, form II (R. rubrum) and form I enzymes (that share
31% identity) have very similar three-dimensional structures
(9). Nonetheless, the importance of robust dimer formation
between two chimeric large subunits cannot be ignored. As Knight et al.
(18) point out, the immense surface area involved in N- and
C-terminal contacts between large subunit dimer pairs in the spinach
enzyme are extremely important to overall holoenzyme stability.
It is also feasible that observed subunit incompatibility is the result
of large differences (34% amino acid sequence identity) between
R. sphaeroides and R. capsulatus small subunit
sequences (27). The N-terminal 14-amino-acid insertion and
C-terminal deletion in the R. capsulatus small subunit
(relative to the R. sphaeroides CbbS sequence) is likely to
effect holoenzyme assembly. Of the small subunit residues present at
large/small subunit interfaces in the spinach holoenzyme, over 50%
came from the first 30 amino acids (of 123 residues) (18).
From these data it is obvious that the subunits of these two enzymes
are too evolutionarily divergent to coalesce into an active, stable,
and properly folded hexadecamic enzyme, using the construction
strategies employed here. Despite previous success in producing a
useful Synechococcus large subunit-diatom small subunit
hybrid that established subunit contributions to various enzymatic
properties (32), the present results are similar to those of
previous in vitro experiments (14) which reported
unsuccessful reconstitution by isolated Chromatium large
subunits and cyanobacterial small subunits. This group and others,
however, were able to successfully assemble functional heterologous
enzymes between more closely related subunits from a variety of
organisms including higher plant small subunits with cyanobacterial
large subunits (1, 2, 12, 22, 32). Perhaps in vivo DNA
shuffling (3) and other in vivo and in vitro methods,
combined with specific genetic selection, which by definition only
results in productive assemblages, will allow for the successful
synthesis of chimeras to provide molecules for the identification of
important functional domains.
Concluding remarks.
High-yield purification of R. capsulatus form I RubisCO has been described. Enzymological
properties determined here firmly establish its green-like nature, and
the results corroborate the suggestion that the genes for this enzyme
were likely transmitted to an ascendant of R. capsulatus via
a horizontal gene transfer event (27). Of particular
importance are the low CO2/O2 substrate specificity, the high affinity for CO2, and the inhibition
by RuBP. Failure to successfully assemble recombinant hybrid
holoenzymes in vivo with subunits from another purple nonsulfur
bacterium further indicate the vast differences of the R. capsulatus protein, as does a preliminary attempt to construct a
chimera between large subunits of these proteins. Finally, observed
similarities in primary structure of the R. capsulatus and
cyanobacterial form I enzymes, and the fact that the
KCO2 is so different for the two
enzymes, suggest that the construction of in vivo chimeras between
these two proteins might be a worthwhile endeavor.
This work was supported by Public Health Service grant GM 24497 from the National Institutes of Health.
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Abstract
Introduction
