(Received for publication, January 22, 1997, and in revised form, February 24, 1997)
From the Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664
The temperature-conditional
photosynthesis-deficient mutant 68-4PP of Chlamydomonas
reinhardtii results from a Leu-290 to Phe substitution in
the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Although this substitution occurs
relatively far from the active site, the mutant enzyme has a reduced
ratio of carboxylation to oxygenation in addition to reduced thermal
stability in vivo and in vitro. In an attempt to understand the role of this region in catalysis,
photosynthesis-competent revertants were selected. Two revertants,
named R96-4C and R96-8E, were found to arise from second-site mutations
that cause V262L and A222T substitutions, respectively. These
intragenic suppressor mutations increase the
CO2/O2 specificity and carboxylation
Vmax back to wild-type values. Based on the
crystal structure of the spinach holoenzyme, Leu-290 is not in van der
Waals contact with either Val-262 or Ala-222. However, all three
residues are located at the bottom of the /
-barrel active site
and may interact with residues of the nuclear encoded small subunits.
It appears that amino acid residues at the interface of large and small
subunits can influence both stability and catalysis.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC
4.1.1.39, Rubisco)1 initiates both
photosynthetic carbon assimilation and photorespiration (see Refs. 1
and 2 for review). The enzyme generates either two molecules of
phosphoglycerate by carboxylation of RuBP or one molecule each of
phosphoglycerate and phosphoglycolate by oxygenation of RuBP. Because
phosphoglycolate enters the fruitless photorespiratory pathway and
leads to the loss of CO2 (see Ref. 3 for review), an
increase in carboxylation or a decrease in oxygenation would likely
improve plant productivity (see Ref. 1 for review). The ratio of
carboxylation to oxygenation at any given concentrations of
CO2 and O2 is defined by the
CO2/O2 specificity factor, = VcKo/VoKc, where
Vc and Vo are the
Vmax values for carboxylation and oxygenation,
and Kc and Ko are the Michaelis constants for CO2 and O2, respectively (4).
Because CO2 and O2 are mutually competitive at
the same active site, the differential stabilization of the
carboxylation and oxygenation transition states ultimately determines
(5, 6).
The Rubisco holoenzyme in the chloroplasts of plants and green algae is
composed of eight copies each of large and small subunits (see Refs. 1
and 2 for review). A family of nuclear rbcS genes encodes
the 15-kDa small subunits, whereas the chloroplast rbcL gene
encodes the 55-kDa large subunits. The small subunit is synthesized as
a 21-kDa precursor in the cytosol and processed to mature form upon
entry into chloroplasts (see Refs. 7 and 8 for review). Holoenzyme
assembly is then facilitated by the action of chloroplast chaperonin 60 (see Ref. 9 for review). The active site of Rubisco is formed at the
interface between the carboxyl-terminal /
-barrel domain of one
large subunit and the amino-terminal domain of a second large subunit
(10-12). Rubisco also requires an additional nuclear encoded protein,
named Rubisco activase, for its activation in vivo (see Ref.
13 for review).
In contrast to directed mutagenesis (2, 14, 15), random screening for
chloroplast rbcL mutations in the green alga Chlamydomonas reinhardtii, followed by genetic selection,
has identified a number of complementing large subunit substitutions that influence (16-19). Because these substitutions are most often
found in the secondary structure elements that comprise the cores of
the
/
-barrel or amino-terminal domains their influence on
is
not readily deduced from the existing x-ray crystal structures (11, 12,
20). In particular, one temperature-conditional mutant strain, named
68-4PP, results from an L290F substitution at the end of
-strand 5 at the bottom of the
/
barrel (21, 22). This substitution reduces
by 13%, and the mutant enzyme has decreased thermal stability at
35 °C in vivo and in vitro (22, 23). Genetic
selection previously identified a nuclear suppressor mutation, named
S52-2B, that restores the thermal stability and
value of the 68-4PP
enzyme back to wild-type levels (23, 24). The S52-2B mutation does not
reside in either of the two rbcS genes (24), and its gene
product affects Rubisco at a posttranslational step (23, 25).
Otherwise, the molecular basis for the S52-2B mutation is not yet
known.
We reasoned that if suppressor mutations could arise in other nuclear
genes or elsewhere in the 68-4PP rbcL gene, analysis of such
suppressors might help to understand the mode of action of the 68-4PP
L290F substitution and, perhaps, the means by which S52-2B acts as a
suppressor. Therefore, additional photosynthesis-competent revertants
were selected from mutant 68-4PP and analyzed. Two of these new
revertants arose from rbcL second-site mutations that change
residues at the interface between large and small subunits at the
bottom of the /
barrel.
C. reinhardtii wild-type 2137 mt+ (26), mutant 68-4PP mt+ (21, 22), and revertant strains are maintained at 25 °C in darkness with 10 mM acetate medium containing 1.5% Bacto-agar (Difco) (26). The temperature-conditional 68-4PP mutant dies on minimal medium in the light at 35 °C but survives on minimal medium at 25 °C in the light or on acetate medium at either 25 or 35 °C in the dark (21). This temperature-conditional photosynthesis deficiency results from an L290F substitution in the Rubisco large subunit (22, 23). For biochemical analysis, cells were grown on a rotary shaker with 500 ml of liquid acetate medium in darkness.
Revertant Selection and Genetic AnalysisIndependent clones
of mutant 68-4PP were used for reversion experiments to ensure the
genetic independence of the revertants (27). Dark-grown mutant cells
were plated on solid minimal medium at a density of 2 × 106 cells/100-mm Petri plate at 35 °C with a light
intensity of 68 microeinsteins m2 s
1 (21,
27). In several reversion experiments, cells were treated with
5-fluoro-2
-deoxyuridine and/or methyl methanesulfonate prior to
selection to increase the recovery of chloroplast mutations (26, 27).
Crosses were performed as described previously (21, 26), and
temperature-conditional, acetate-requiring progeny were scored by
replica-plating dark-grown (25 °C) tetrads to minimal medium in the
light at 35 °C.
Isolation of total DNA, amplification of rbcL by the polymerase chain reaction, subsequent gene cloning of the amplified products, and DNA sequencing were performed as described previously (18). Several independently cloned rbcL genes were partially sequenced to confirm the assignment of mutations, and at least one complete rbcL gene from each revertant was sequenced to ensure that only the expected mutations were present.
Biochemical AnalysisAbout 2 × 109 cells
were harvested by centrifugation and sonicated in 1 mM
dithiothreitol, 10 mM MgCl2, 10 mM
NaHCO3, and 50 mM
N,N-bis(2-hydroxyethyl)glycine, pH 8.0, at
0 °C for 3 min. Protein was quantified by the method of Bradford
(28). The cell extracts were fractionated by SDS-polyacrylamide gel
electrophoresis (29), and Western analysis was performed as described
previously (25). Rubisco holoenzyme was purified from cell extracts by sucrose gradient centrifugation (30). RuBP carboxylase activity was
measured as the incorporation of acid-stable 14C from
NaH14CO3. was determined by the
simultaneous measurement of the carboxylase and oxygenase activities of
purified Rubisco (20 µg/reaction) with 200 µM
[1-3H]RuBP (7.2 Ci/mol) and 2 mM
NaH14CO3 (5.0 Ci/mol) in 30-min reactions at
25 °C (27, 31). Synthesis and purification of
[1-3H]RuBP were performed according to the methods of
Kuehn and Hsu (32). Other kinetic constants of the purified enzymes
were determined as described previously (22).
In
a previous study (24), photosynthesis-competent revertants were
recovered at a frequency of 7 × 109 cells by
directly plating rbcL mutant 68-4PP
mt+ on minimal medium in the light at 35 °C.
In the present study, revertants of 68-4PP mt+
were recovered spontaneously (at a frequency of 5 × 10
9) or they were recovered after methyl methanesulfonate
or 5-fluoro-2
-deoxyuridine/methyl methanesulfonate treatment (at a
frequency of 1 × 10
8) as a means for increasing the
number of potential suppressor mutations (17, 26, 30). Genetic analysis
of 11 revertants revealed that two of them, named R96-4C and R96-8E,
were inherited in a uniparental pattern indicative of mutations in
chloroplast DNA. Both of these strains were recovered following methyl
methanesulfonate treatment. When the rbcL genes from wild
type, R96-4C, and R96-8E were cloned, sequenced, and compared, both of
the revertants were found to arise from intragenic suppression. In
addition to the original 68-4PP (L290F) mutation, the revertant R96-4C
rbcL gene also contained a G to T transversion mutation that
would change Val (GTA) to Leu (TTA) at large subunit residue 262. Because this mutation eliminated an RsaI restriction site,
the assignment of the mutation was further verified by restriction
analysis of revertant R96-4C DNA. DNA sequencing of the R96-8E
rbcL gene revealed a G to A transition mutation that would
change Ala (GCT) to Thr (ACT) at large subunit residue 222. Thus, the
L290F substitution caused by the original mutation can be complemented
by either a V262L or A222T substitution. Because either substitution
could restore photosynthetic growth of the 68-4PP mutant at the
35 °C restrictive temperature, we assumed that both substitutions
must affect Rubisco catalysis or stability.
When extracts of 35 °C-grown wild-type, mutant, and revertant cells were fractionated on sucrose gradients, the revertants were found to have a modest increase in the amount of Rubisco holoenzyme relative to that of the 68-4PP mutant (Table I). However, the revertants had only about 30% of the wild-type level of holoenzyme when grown at 35 °C, and when grown at 25 °C they had less holoenzyme than the 68-4PP mutant (Table I). To confirm that the levels of extractable holoenzyme represented the levels of holoenzyme in vivo, extracts were fractionated on SDS-polyacrylamide gels and analyzed by immunoblotting (data not shown). The V262L and A222T substitutions may complement the L290F mutant substitution by partially restoring Rubisco thermal stability in vivo at 35 °C, but this "improvement" is associated with a decrease in holoenzyme stability at the 25 °C permissive temperature (Table I).
|
The ratio between Rubisco carboxylase activities measured at limiting CO2 (0.53 mM NaHCO3) under 100% N2 and 100% O2 is a function of the Kc and Ko kinetic constants (22, 24). When these ratios were determined for purified Rubisco, wild-type and 68-4PP (L290F) mutant enzymes had values of 3.0 and 2.4, respectively. The R96-4C (L290F/V262L) and R96-8E (L290F/A222T) revertant enzymes had N2/O2 ratio values of 2.2 and 2.8, respectively, indicating that these enzymes had kinetic properties different from either the wild-type or 68-4PP mutant enzyme.
The 68-4PP (L290F) mutant enzyme is known to have an value lower
than that of the wild-type enzyme (22, 24). Detailed biochemical
analysis of purified Rubisco confirmed that the 68-4PP enzyme has a
17% decrease in
and revealed that the revertant enzymes have
values restored to the wild-type value (Table II). The
improved
values of both revertant enzymes arise from increases in
Vc, Vc/Kc, and
Ko/Kc relative to the original
68-4PP mutant enzyme (Table II). However, neither enzyme has a
Vc or Vc/Kc
value as good as that of the wild-type enzyme. With regard to the
revertant R96-4C (L290F/V262L) enzyme, the improved
occurs despite
an increase in Kc and decrease in
Vc/Vo relative to the values of
the 68-4PP (L290F) mutant enzyme.
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Mutant 68-4PP Rubisco has a decreased (22) and reduced thermal
stability in vivo and in vitro (22, 23). These
enzyme defects arise from an L290F substitution in the
chloroplast-encoded Rubisco large subunit (22, 24). According to the
crystal structure of spinach Rubisco (10, 12), Leu-290 is the first
residue of
-strand 5 at the bottom of the
/
barrel. As such,
it is relatively far from the active site residues that coordinate with the transition-state analog carboxyarabinitol 1,5-bisphosphate (10,
12). It was previously suggested (1) that the L290F substitution may
disrupt the hydrophobic core of the
/
barrel by affecting a
hydrogen bond network that extends from Glu-168 (at the bottom of the
/
barrel) to active-site His-327 (at the base of flexible loop 6)
(10).
Because the 68-4PP (L290F) mutation gives rise to a
temperature-conditional, acetate-requiring phenotype in vivo
(21, 22), it was possible to select for complementing mutations at the
35 °C restrictive temperature. Past genetic studies with
Chlamydomonas Rubisco have identified compensatory
substitutions at residues that are often in van der Waals contact with
the original mutant residues (16-18). However, second-site
substitutions that complement L290F were found to be relatively
distant. Either a V262L or A222T substitution produces a moderate
increase in the amount of L290F mutant Rubisco at 35 °C (Table I)
and, more significantly, restores to the wild-type value (Table
II). Because the V262L and A222T substitutions both increase the volume
of the affected residues, it seems unlikely that they compensate for
the increase in size caused by the original L290F mutant substitution.
Furthermore, based on the x-ray crystal structure of spinach Rubisco
(10, 12), neither of these phylogenetically conserved residues resides in the Rubisco active site. Ala-222 (in the middle of
-helix 2) is
more than 10 Å away from the atoms of Leu-290. Whereas Val-262 (below
-strand 4) appears to be close to Leu-290 (bottom of
-strand 5),
the side chains of these two residues are oriented away from each
other. Instead, Val-262 is in van der Waals contact with Ala-222 (10,
12). These "long-distance" interactions relative to residue 290 are
interesting, especially considering that residues 222, 262, and 290 are
close to residues of the Rubisco small subunit (10, 12).
In spinach Rubisco, a small subunit hairpin loop (residues 46-67
flanked by -strands A and B) is in close contact with large subunit
residues at the bottom of the
/
barrel (10). Cyanobacterial Rubisco lacks 12 residues of this loop (residues 52-63), whereas Chlamydomonas Rubisco contains 6 additional residues (see
Ref. 1 for review). Because
values also diverge among these
species, it is interesting to consider whether the small subunit
hairpin loop might contribute to the enhanced catalytic efficiency of eukaryotic Rubisco (10, 20). With regard to the crystal structure of
spinach Rubisco (10, 12) and as shown in Fig. 1, the
L290F substitution could place residue 290 in van der Waals contact with small subunit residues Gly-60 and Tyr-62 (Leu-66 and Tyr-68 in
Chlamydomonas Rubisco, respectively). Furthermore, the
backbone atoms of large subunit Val-262 are likely to be in van der
Waals contact with the C
, C
, and
C
atoms of the spinach small subunit Pro-59 (Cys-65 in
the Chlamydomonas enzyme), which is located at the tip of
the small subunit hairpin loop (10) (Fig. 1). The C
atom
of large subunit Ala-222 is also in van der Waals contact with one of
the C
atoms of spinach small subunit Tyr-61 (Tyr-67 in
the Chlamydomonas enzyme). However, this tyrosyl residue
resides in a second neighboring small subunit (10) (Fig. 1). In
conclusion, it seems possible that the V262L and A222T large subunit
substitutions could complement the L290F substitution via interactions
transmitted through the Rubisco small subunit (Fig. 1). Perhaps these
interactions at the interface between large and small subunits
contribute to both the structural stability (Table I) and catalytic
efficiency (Table II) of Rubisco.
Directed mutagenesis of pea rbcS followed by in vitro synthesis and transport into isolated chloroplasts indicated that Arg-53 in the small subunit hairpin loop is required for holoenzyme assembly (33). In spinach Rubisco (10, 12) this arginyl residue (Arg-59 in the Chlamydomonas enzyme) hydrogen bonds with large subunit Tyr-226, which is also in van der Waals contact with the side chain atoms of Ala-222 and Val-262. Such observations further support the idea that substitutions at Ala-222 and Val-262 may also influence the interactions between large and small subunits.
When a hybrid Rubisco enzyme comprised of cyanobacterial large subunits
and diatom small subunits was expressed in Escherichia coli,
it was found to have an value intermediate to the
values of the
native cyanobacterial and diatom holoenzymes (34). Thus, even though
the hybrid enzyme had substantial decreases in Vc and Vc/Kc (34), these results
indicate that small subunits can contribute to the catalytic efficiency
of Rubisco. Considering that substitutions at large subunit residues
290, 222, and 262 can influence
(Table II), perhaps the nearby
small subunit residues (Fig. 1) could also play a role in determining the catalytic efficiency of Rubisco.
Most of the revertants recovered from mutant 68-4PP in this and a previous study (24) arose from nuclear suppressor mutations. One of these revertants has been analyzed in detail (23, 24), but the nuclear suppressor mutation does not reside in either of the two rbcS genes (24). Nonetheless, the idea that small subunits can act as a bridge between L290F and the complementing V262L and A222T substitutions would imply that substitutions in the small subunit may also complement the original 68-4PP (L290F) mutation. Further study of the existing nuclear suppressors will test this hypothesis.
Because the L290F, A222T, and V262L substitutions can influence Rubisco
(Table II) and may do so by influencing the structure of the large
subunit/small subunit interface (Fig. 1), it would be interesting to
examine the effect of nearby small subunit substitutions on Rubisco
catalysis. However, it has not been possible to create such
substitutions via directed mutagenesis because prokaryotic Rubisco
enzymes lack either the small subunit or the small subunit hairpin loop
(see Ref. 1 for review), and eukaryotes contain a family of
rbcS genes that precludes transformation of mutant genes
(see Refs. 1 and 8 for review). Only recently has a mutant strain of
Chlamydomonas been recovered that lacks the rbcS
gene family (35). Because the rbcS deletion mutant can be
transformed with a single rbcS gene (35), it may soon be possible to directly investigate the potential role of small subunit residues 65, 66, 67, and 68 in Rubisco catalysis.
We thank Carolyn M. O'Brien for performing Western analysis. Rubisco antibody was generously supplied by Dr. Raymond Chollet.