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Archives of Biochemistry and Biophysics 414 (2003) 150–158
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The life of ribulose 1,5-bisphosphate
carboxylase/oxygenase—posttranslational facts and mysteriesq
Robert L. Houtza and Archie R. Portis Jr.b,*
a
b
Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, N322D Agricultural Science Center North,
University of Kentucky, Lexington, KY 40546-0091, USA
Photosynthesis Research Unit, Agricultural Research Service, United States Department of Agriculture and Department of Crop Sciences
and Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA
Received 13 December 2002, and in revised form 26 February 2003
Abstract
The life of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), from gene to protein to irreplaceable component of
photosynthetic CO2 assimilation, has successfully served as a model for a number of essential cellular processes centered on protein
chemistry and amino acid modifications. Once translated, the two subunits of Rubisco undergo a myriad of co- and posttranslational modifications accompanied by constant interactions with structurally modifying enzymes. Even after final assembly, the
essential role played by Rubisco in photosynthetic CO2 assimilation is dependent on continuous conformation modifications by
Rubisco activase. Rubisco is also continuously assaulted by various environmental factors, resulting in its turnover and degradation
by processes that appear to be enhanced during plant senescence.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase; Posttranslational; Methylation; Protein folding; Ribulose 1,5-bisphosphate; Activase;
Oxidation
Recognized for decades as a unique catalyst and
limiting factor in photosynthetic CO2 assimilation, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)1 has been the subject of numerous intense
scientific studies. This interest has resulted in many
pioneering investigations that now serve as models for
a variety of cellular mechanisms that are not restricted
to plant photosynthetic processes. In this review, we
focus on co- and posttranslational modifications that
accompany the biochemical life of this enzyme. The
diversity of these modifications as they relate to the
synthesis, assembly, regulation, and degradation of
q
This work was supported by U.S. Department of Energy Grants
DEFG02-92ER20075 (R.L.H.) and DEAIG2-97ER20268 (A.R.P.).
*
Corresponding author. Fax: 1-217-244-4419.
E-mail address: [email protected] (A.R. Portis Jr.).
1
Abbreviations used: DEF, peptide deformylases; HMT, histone
methyltransferase; LS, large subunit; LSMT, LS e N -methyltransferase;
PMSR, peptide Met sulfoxide reductase; SS, small subunit; SSMT, SS
a
N -methyltransferase; Rubisco, ribulose 1,5-bisphosphate carboxylase/
oxygense; RuBP, ribulose-1,5-bisphosphate.
Rubisco are summarized in Fig. 1. Many of these
structural and conformational changes have been
shown to be instrumental in the regulation and assembly of Rubisco but, more importantly, several remain as largely unexplored areas with potential to
increase the body of knowledge about this crucial
carbon-fixing enzyme.
Amino acid modifications
Both the large subunit (LS) and the small subunit
(SS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) are extensively modified either during
and/or after translation. Evidence for these amino acid
modifications comes primarily from structural analyses
of the mature forms of the polypeptides and subsequent
comparison with the known DNA sequences. In some
cases, this evidence is corroborated by the characterization of specific enzymes responsible for these modifications.
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0003-9861(03)00122-X
R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158
Fig. 1. Schematic summary of various co- and posttranslational
modifications of Rubisco that are currently known to occur during the
three phases of its life cycle. Some modifications are species specific.
SS modifications
Prior to import into the chloroplast, the N-terminal
transit sequence of the SS is phosphorylated (probably
at Ser-21) as part of a process that partially regulates
import [1]. During or immediately after import, the
transit sequence is removed by a stromal processing
peptidase in what appears to be a two-step proteolytic
process [2–6]. The final processing exposes a conserved
Met residue, which is methylated to form an a N -methylmethionine [7,8]. This is a widespread modification
found in the SS of Rubiscos from a number of species
[7,9–11]. The enzyme responsible for this modification,
Rubisco SS a N -methyltransferase (SSMT) [11], is related
to the LS e N -methyltransferase (LSMT) responsible for
the methylation of Lys-14 in the LS. Although the
functional significance of this modification is unknown,
it would provide a mechanism for differentiating between N-terminal Met residues on imported proteins
and proteins translated in the chloroplast and may
provide protection against aminopeptidases.
LS modifications
The N-terminal residue of the LS of Rubisco in all
higher-plant species examined to date is acetylated Pro-3
[12–14]. This region of Rubisco is not discernible by Xray crystallography, and identification has been exclusively by mass spectroscopy of proteolytically removed
N-terminal fragments. Comparison of the N termini of
the LS with the predicted translation product from the
DNA sequence clearly indicates a number of co- and
posttranslational modifications. First, protein translation in the chloroplast is initiated with N -formylmethionine [15], and the LS of Rubisco likely begins with this
residue. During translation deformylation of N -formyl-
151
methionine occurs by chloroplast-specific peptide
deformylases (DEF) (EC 3.5.1.88) [16–18]. As in prokaryotic organisms, this is an essential process [16,17,19]
and one upon which all subsequent N-terminal processing is dependent. A biochemical characterization
and determination of polypeptide substrate specificity of
chloroplast DEFs has been reported. The data suggest
that the two chloroplast-localized DEFs have different
polypeptide substrate specificities and kinetic parameters [17,19,20].
Shortly after removal of the N -formyl group, Met-1 is
removed, probably as a result of N-terminal processing
by chloroplast-specific Met aminopeptidases [16,18].
Transcripts that code for such peptidases with predicted
chloroplast-targeting sequences have been reported in
Arabidopsis, but direct detection and characterization of
these enzymes remains to be shown [18]. Although
acetylated Ser-2 was reported earlier as the N-terminal
residue of the LS [21], the structural identification of the
N terminus of the LS of Rubisco from a wide range of
species suggests that further N-terminal processing results in the removal of Ser-2 and subsequent acetylation
of Pro-3 [13]. Whereas amino peptidases have been reported in the chloroplast, none have been described with
specificity for N-terminal Ser residues [22]. How Ser-2 is
removed remains to be described. Alternatively, removal
of Met–Ser as a dipeptide could occur. The remaining
N-terminal Pro residue is acetylated and this could
provide protection against proteolytic attack since Prospecific iminopeptidases have been reported in both
leaves and chloroplasts [22,23]. Na -acetylation is one of
the most common cotranslational protein modifications,
perhaps occurring on 80% of all proteins [24–26]. Genes
encoding N a -acetyltransferases have been sequenced
and characterized from Saccharomyces cerevisiae [27–
30] and Trypanosoma brucei [31]. At least in two reports,
the activity of these enzymes has been shown to be essential. To date, however, no chloroplast-localized N a acetyltransferases have been described or annotated in
plant genome sequences.
In some but not all plant species, the LS is additionally modified by trimethylation of the e-amino group
of Lys-14 to form a trimethyllysine residue [12,13]. The
modification is common in the LS of Rubisco from
Solanaceae and Cucurbitaceae species, but there is still
not enough information to establish any definitive species correlation with the absence or presence of a trimethyllysine residue. The temporal nature of this
modification is not known, but studies have clearly
demonstrated that this modification can occur posttranslationally even after holoenzyme assembly [32,33].
The enzyme responsible for this modification is a Rubisco LSMT (EC 2.1.1.127) [34–36]. Although many
different proteins contain trimethyllysine residues, each
is associated with a specific protein e N -methyltransferase
[37]. The protein e N -methyltransferase responsible for
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the methylation of Lys-14 in the LS of Rubisco has been
thoroughly characterized. A high-resolution crystal
structure for pea Rubisco LSMT which identified a
novel bipolar arrangement of substrate binding sites was
recently obtained. One binding site has specificity for the
N-terminal region of the LS of Rubisco and the other
for S-adenosylmethionine, with a pore through which
methyl group transfer occurs connecting the two sites
[38]. Models describing the docking of Rubisco LSMT
to Rubisco suggest that extensive regions of Rubisco
LSMT are involved in polypeptide recognition and that
the molar stoichiometry of Rubisco LSMT binding to
Rubisco is 4 to 1 (Fig. 2). The substrate binding sites are
located in a conserved polypeptide sequence (SET domain) with secondary structural homology to a large
number of highly evolutionarily conserved proteins
known to influence gene expression [39,40]. Many of
these proteins have been recently identified as histone
methyltransferases (HMTs) [41–44]. Moreover, the
mechanism for HMT influence on gene expression led to
the identification of proteins that are recruited to histone methylation sites through a specific binding domain
for trimethyllysine residues known as the chromodomain [45–47]. Since the functional significance of trimethyllysine-14 in the LS of Rubisco remains an enigma,
perhaps these recent advances in discerning the functional significance of Lys methylation in histones will
provide clues for identification of the functional significance of trimethyllysine residues in Rubisco and may
identify chloroplast proteins recruited to the N-terminal
region of Rubisco.
Recently the structural determination of Chlamydomonas Rubisco revealed the presence of several other
amino acid modifications in the LS [9,10]. Both Pro
residues 151 and 104 were found as 4-hydroxyproline
residues and Cys residues 256 and 369 as S (gamma)methylcysteine residues. These modifications have not
been observed in Rubiscos from other species and may
be unique to algal forms of Rubisco.
Probably the most dynamic amino acid modification
in the LS is the carbamylation of the e-amino group of a
highly conserved active-site Lys residue [48,49]. The
carbamylation of Lys-201 (Lys-191 in Rhodospirillum
rubrum) results in the formation of a metal binding site
and, after metal addition, a catalytically competent enzyme. Additionally, the carbamate formed on Lys-201
has been proposed as the essential base responsible for
proton abstraction from the C3 position of ribulose 1,5bisphosphate (RuBP) and subsequent ene-diol formation [50]. While carbamylation of the isolated enzyme is
a facile and spontaneous process in vitro, the carbamylation level of the enzyme in vivo is determined by the
concentrations of various effector ligands that bind to
the active site and the interaction of Rubisco with Rubisco activase (discussed below).
Clearly the LS and SS of Rubisco are subject to a
rather large number of both co- and posttranslational
modifications. Given the abundance of Rubisco in the
biological world, these amino acid modifications may
also represent the most abundant forms of these modified amino acids.
Folding and assembly
Fig. 2. Model indicating the probable binding between pea Rubisco
LSMT and spinach Rubisco. The lower half of the spinach Rubisco
holoenzyme structure has been removed, leaving the top octamer of
four LSs (light green) and four SSs (dark green) to aid in visualization
of Rubisco LSMT. Rubisco LSMT is positioned favorably for Lys-14
(gold) methylation, and the AdoMet binding site is occupied by Sadenosyl homocysteine (red). Reprinted from [38] with permission
from Elsevier Science.
In prokaryotic organisms, newly translated proteins
interact in an ATP-dependent reaction with a welldescribed set of chaperones known as DnaK, DnaJ, and
GrpE prior to final assembly by GroEL/GroES chaperonins [51]. The model for this interaction involves the
association of DnaJ proteins with newly translated
polypeptides, recruitment of DnaK proteins to the same
polypeptide, and final formation of a tertiary complex
with GrpE. These complexes prevent undesirable polypeptide interactions and potential aggregation. Chloroplast homologs of DnaK [52–54], DnaJ [55], and GrpE
[55,56] have been described. Whereas the LS of Rubisco
has not been found in direct association with these
chaperones, other polypeptides that must be imported
into the chloroplast have [57,58] including the transit
peptide of the SS [59]. Moreover, the SS transit peptide
has been shown to interact directly with chloroplast
Hsp70 during import and to contain a recognition domain for Hsp70 [59–61]. The complexes formed between
the newly translated polypeptides and the DnaK/DnaJ/
R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158
GrpE complexes represent the first step in cooperative
protein folding that ends with the release from the
DnaK/DnaJ/GrpE complexes to the GroEL/GroES
chaperonins. A similar set of reactions probably operates
in chloroplasts but with regard to the LS of Rubisco,
more information exists at the level of GroEL/GroES
folding than at the earlier steps in protein folding.
Evidence for chaperonin-mediated protein folding in
the chloroplast was found over 2 decades ago when
studies revealed that newly synthesized LSs were transiently associated with a large multimeric complex [62]
prior to appearance in the holoenzyme through an ATPdependent reaction [63]. It is now well documented that
the assembly of Rubisco requires chloroplast homologs
of the GroEL and GroES chaperonins [52,64–67] and
that many if not all chloroplast-imported proteins interact with these proteins [68]. However, the absence of
an in vitro or bacterial expression system for the assembly of higher-plant Rubisco is evidence of the unique
nature of the chloroplast chaperonins and the limitations of our current understanding. The GroEL homologs in the chloroplast are represented by two distinct a
and b chaperonin 60s with only 50% sequence homology between them [69]. The exact stoichiometry and
arrangement of the a and b subunits in the functional
tetradecamer is not known, but tetradecamers composed
of only b subunits are significantly less effective in refolding dimeric R. rubrum Rubisco in vitro [70]. The
chloroplast GroES homologs are as unique as the a and
b chaperonins. Chloroplast chaperonin 10 is much larger than other GroES homologs and is comparable to a
fusion between two GroES peptides [71,72]. Protein
folding by chloroplast chaperonin 60 tetradecamers requires chaperonin 10, but this requirement can be met
by mitochondrial or mouse homologs [70]. The unique
nature of the chloroplast chaperonins suggests that there
may be protein substrate specificity for chloroplast
proteins [70,73], and exploitation of recombinantly expressed forms of the chloroplast chaperonins could lead
to the identification of conditions capable of catalyzing
the folding and assembly of Rubisco in bacterial or in
vitro systems. Such a development would represent a
significant advancement in efforts to exploit molecular
genetic engineering alterations aimed at increasing the
efficiency of Rubisco. However, there may be other requirements for the successful folding and assembly of
hexadecameric Rubisco, in particular the activity of
other enzymes known to influence proper protein folding, such as peptide disulfide isomerase [74,75] and
peptidyl-prolyl cis-trans isomerase [76–78].
Ligand binding and allostery
Once properly assembled, the activity of LS8 SS8
Rubiscos can be modified by interaction with the sub-
153
strate RuBP and a wide variety of other effector ligands;
phosphoglyceric acid and 2-carboxyarabinitol 1-phosphate are particularly important. The interactions are
very complex because (a) both the carbamylated and the
uncarbamylated forms of the enzyme are involved, (b)
binding can induce a conformational change in the enzyme that closes off the active site, offering a potential
for hysteresis, and (c) the two active sites in each dimer
of large subunits are composed of residues at the Cterminal domain of one subunit and the N-terminal
domain of the other subunit. Thus, there is the opportunity for allostery within each dimer and even perhaps
between dimers.
To a first approximation, the effects of various effectors on Rubisco are largely accounted for by a simple
kinetic model in which they bind to both carbamylated
and uncarbamylated Rubisco sites in a competitive
manner with RuBP at the active site [79]. This model can
account for most ‘‘positive’’ and ‘‘negative’’ effectors
and order of addition and dilution effects with in vitro
assays reported in the early literature. Under steady
state conditions with this model, all effectors will increase the Km for RuBP [80]. In vivo, the situation is
complicated by the high concentration of Rubisco sites
relative to the concentration of RuBP and various effectors, but equations for the activity of Rubisco under
these conditions have been derived [81].
However, the binding of RuBP and various effector
ligands is further complicated by a tight binding mode at
the active site. The large number of Rubisco crystal
structures provides a molecular rationale for the tight
binding mode because they form two groups corresponding to either an open or a closed active site [82].
The active site is located at the face of an a=b barrel
formed by the C-terminal domain of the LS. The loops
connecting the b strands to the a helices contribute the
majority of the residues at the active site, but some are
also contributed by the N-terminal domain on the
neighboring LS. In the closed structures, the ligand at
the active site is sequestered from the solvent by loop-6
and the C terminus. Compared to the open structures,
the P1 to P2 phosphate distance is reduced in the closed
structures, and the N-terminal domain in the neighboring LS is positioned closer, with little change in the
positions of the a=b barrels. When the active site is
carbamylated and occupied by RuBP, C–C cleavage
during catalysis appears to be sufficient to reopen the
site, but otherwise release of the tightly bound ligand
associated with the ‘‘closed’’ structures requires movement of these domains.
A reduction in the catalytic activity of Rubisco due to
tight RuBP binding to the uncarbamylated enzyme
[83,84] and 2-carboxyarabinitol 1-phosphate binding to
the carbamylated enzyme have been observed in vivo
[85,86]. In vitro, activity of the enzyme also slowly declines in a process referred to as fallover [87]. Fallover
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has been attributed to the formation of a tight binding
inhibitor during catalysis [88]. However this early
characterization of fallover has been complicated by the
subsequent observations that (a) RuBP is oxidized to
form tight binding inhibitors [89], which may also occur
in vivo [90,91], and (b) the rate of fallover is increased
when catalysis is conducted at higher temperatures [92],
conditions also favoring RuBP oxidation. Further
complicating the issue is the proposal that RuBP binding to additional ‘‘allosteric’’ sites participates in fallover and the observed reduction in Rubisco activity
[93,94]. The relative importance of these multiple fallover processes in contributing to changes in Rubisco
activity in vivo remains to be clearly established.
It should be noted that steady state leaf photosynthesis can be successfully predicted over a wide range of
conditions from knowledge of the electron transport
limitations and kinetic parameters of Rubisco with respect to CO2 and O2 without knowledge of the RuBP
concentration or concentrations of known effectors [95].
However, deficiencies in our understanding of Rubisco
regulation sometimes become apparent if measured
rates of photosynthesis are compared with estimates
derived from measurements of extractable Rubisco activity and the levels of RuBP, phosphoglyceric acid, and
other metabolites in the leaves [96]. A particularly
striking example was obtained in comparisons of RuBP
and phosphoglyceric acid levels during a diurnal light
cycle using growth chamber- and field-grown plants [97].
The observed response of photosynthesis to these metabolites could be simulated in vitro by reciprocally
varying the concentrations of RuBP, phosphoglyceric
acid, and inorganic phosphate, but the response did not
follow that predicted from the modified Michaelis–
Menten Rubisco kinetic model. The problem is that
Rubisco activity does not saturate at high RuBP concentrations and thus cannot be modeled by Michaelis–
Menton kinetics at millimolar concentrations of this
substrate [98]. The observed RuBP kinetics can be
modeled assuming half-of-the-sites reactivity [99] in a
dimer of the large subunits in which RuBP binding to
one site increases both the Km and the Vmax for catalysis
at the second site (J.C. Serviates and A.R. Portis, unpublished).
Allostery between the two active sites in a dimer, and
possibly between dimers, is also indicated by other observations. The binding of RuBP, the transition state
analogue 2-carboxyarabinitol 1,5-bisphosphate, and
other sugar phosphates to both the carbamylated and
the uncarbamylated enzyme can exhibit negative cooperativity and saturate before all eight sites are filled
with a tightly bound ligand ([100–103]; E.M. Larson and
A.R. Portis, unpublished).
Early measurements of ligand binding to Rubisco
indicated that there was only one site, identical to the
active site. Subsequently, evidence for another, regula-
tory, site has been reported by one research group for
RuBP [104], 2-carboxyarabinitol 1,5-bisphosphate [105],
inorganic phosphate [106], and 6-phosphogluconate
[107]. Crystal structures of Rubisco in the presence of a
variety of ligands have not provided any evidence for the
location of this site. However, in one of the ‘‘open’’
Rubisco structures, inorganic phosphate was recently
observed to bind outside of the active site in a ‘‘latch’’
site normally occupied by Asp-473 in the closed Rubisco
structures [82]. Two widely separated residues (Lys-21
and Lys-305) appear to be indirectly involved with the
allosteric site and with the occurrence of a decline in
Rubisco activity when assayed in vitro [108]. These
residues have also been associated with microheterogeneity among the LSs in a crystal structure of the
carbamylated spinach enzyme complexed with 2carboxyarabinitol 1,5-bisphosphate [109]. However, no
evidence for microheterogeneity was observed in another high-resolution structural determination of this
same complex [110]. Thus, at the present time, the
physiological significance of the regulatory ligand
binding sites and LS structural microheterogeneity is not
clear.
Activase
Rubisco activase appears to catalyze a rather unusual
posttranslational modification of Rubisco, necessitated
by the conformational changes associated with the tight
binding mode of ligands such as RuBP with the uncarbamylated enzyme and 2-carboxyarabinitol 1-phosphate with the carbamylated enzyme. To facilitate the
release of these tightly bound sugar phosphates and
rapidly restore activity to Rubisco, plants utilize Rubisco activase to reopen the catalytic site [111]. Rubisco
activase is a member of the AAAþ (ATPases associated
with diverse cellular activities) protein family that constitutes a wide variety of proteins with chaperone-like
functions, typically involving the disruption of molecular and macromolecular structures [112].
The three-dimensional molecular structure of activase
is not known, but the AAAþ module appears to have a
conserved fold [112]. Consistent with many AAAþ
proteins in which the AAAþ module is linked covalently
to domains that determine the actual cellular function,
residues at both the N and the C termina outside of the
AAAþ module in Rubisco activase appear to interact
with Rubisco [113,114]. A region on the LS of Rubisco,
which is adjacent to the active site, has been identified
using site-directed mutagenesis to change the specificity
between Chlamydomonas Rubisco and heterologous
Rubisco activases [115,116]. AAAþ proteins usually
participate in macromolecular complexes composed of
several AAAþ protein subunits, often assembled in rings
where the ATPase activity appears to act as a motor
R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158
driving inter- and intramolecular movements [112].
Similarly, the activase subunits are highly self-associating, increasing in both ATPase and Rubisco activation
with the extent of oligomerization [117,118]. Based on
AAAþ protein analogies, the ‘‘open’’ and ‘‘closed’’
Rubisco structures, and a large body of research on the
activase, a model for the mechanism of the activase and
its interaction with Rubisco has been proposed [111].
Rubisco activase usually consists of two isoforms
generated by alternative splicing of a pre-mRNA [119–
121]. The activity of the activase is regulated by the
ADP/ATP ratio and the response is modified by redox
changes in the larger isoform that are mediated by thioredoxin-f [122]. These features allow the activity of
Rubisco to be regulated by the activase in response to
light intensity [123] and down-regulated in response to
triose phosphate use limitations [124] associated with
inadequate sinks. However, some species lack a larger
isoform, and the exact mode of activase/Rubisco regulation in these species remains unclear [125]. A detailed
review of the activase is available [111].
Oxidation
When plants are placed under various types of environmental stress, a common response is accelerated senescence and a loss of Rubisco protein. The idea that
Rubisco degradation during this process is dependent
upon oxidative modification of the protein emerged
quite early [126] and recently has been reiterated
[127,128]. However the molecular characterization of
the modifications in Rubisco when plants are placed
under stress has been difficult and this may be due to
rapid turnover of the modified protein [129]. Several
types of stresses that result in Rubisco degradation are
known: UV radiation, ozone, nutrient limitations, and
other stresses (e.g., osmotic or high light) that are
known to generate active oxygen species (hydrogen
peroxide, superoxide, and hydroxide radicals) in the
chloroplasts and/or be associated with an accelerated
senescence.
UV radiation causes a covalent nondisulfide crosslinkage of the LS and SS (65- to 66-kDa product)
attributed to Trp photolysis [130] followed by polymerization into very large molecular-mass aggregates
[131,132]. The specific Trp residues involved have not
been identified.
The specific modifications in Rubisco associated with
the exposure of intact plants to ozone are not as clear.
The oxidation and aggregation of Rubisco induced by
ozone exposures in vitro with the isolated enzyme or
isolated chloroplasts could not be detected in intact
tissue [133]. Due to its high reactivity, it is unlikely that
ozone penetrates into the chloroplast and oxidizes Rubisco directly [134], but ozone can result in the forma-
155
tion of active oxygen species that could then oxidize
Rubisco [133]. Carbonyl derivatives on the side chains
of His, Arg, Lys, and Pro are the typical products of
protein oxidation [135]. Carbonyl residues in numerous
Phaseolus vulgaris proteins, including the Rubisco SS,
were recently detected after chronic ozone stress [136].
Active oxygen species have also been shown to cause
Rubisco breakdown into distinct fragments in isolated
chloroplasts [137,138]. Active oxygen treatment of Rubisco in vitro results in modifications that allow Rubisco
to become a substrate for stromal proteases [139]. The
hydroxl radical has been implicated as a major player in
this process [140] and Gly-329 is a key residue, which
can be directly attacked by the hydroxyl radical to cause
fragmentation [141].
Another residue associated with environmental stress,
oxidation, and increased degradation of Rubisco is Cys
[142–146]. Cys oxidation also primes Rubisco for proteolysis in vitro [143,147,148]. The formation of mixed
disulfides [128] and intermolecular cross-linking of the
large subunits via Cys-247 [144] may be important in
Rubisco degradation in vivo. Site-directed mutagenesis
in Chlamydomonas was recently used to investigate the
role of Cys-172, which may form a disulfide with Cys192, in Rubisco stability. A substitution of Cys-172 with
Ser resulted in an enzyme that was less stable to high
temperatures in vitro, but was more resistant to protease
digestion at low redox potentials [149]. However, more
significantly, the stability of the enzyme in vivo to oxidative (H2 O2 ) and osmotic (mannitol) stress was increased. Site-directed mutagenesis is clearly a powerful
technique to further investigate the role of specific Cys
or other residues in Rubisco stability under various
oxidative stresses.
One of the amino acids most susceptible to oxidation
is Met, which under physiological conditions leads to
the formation of Met sulfoxide residues [150]. The
presence of Met sulfoxide residues in polypeptides can
result in functional inactivation [151,152] and targeting
for proteolytic degradation [153]. However, in all organisms examined thus far, a thioredoxin-dependent
enzyme, peptide Met sulfoxide reductase (PMSR), is
capable of catalyzing the reduction of Met sulfoxide
residues back to Met and restoration of polypeptide
function [154]. A plastidal form of PMSR has been
cloned from Arabidopsis and is localized in chloroplasts
[155,156]. A recent report confirms the ability of chloroplastic PMSR to restore functional competency to a
chloroplastic heat shock protein (Hsp21) inactivated by
oxidation of Met residues [157]. An interesting and
significant hypothesis with regard to the cyclic oxidation
and reduction of Met residues in proteins by PMSR is
that this is a cellular mechanism especially well suited to
an antioxidant defense mechanism [150,158–160]. With
regard to the chloroplast, the high concentration of
Rubisco with 96 Met residues per holoenzyme (spinach
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R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158
enzyme) suggests that the continuous oxidation and reduction of methionine residues could conceivably result
in a rather large consumption of oxidative metabolites.
The role that cyclical oxidation and reduction of Met
residues in Rubisco plays in turnover or activity remains
to be elucidated. Finally, a very recent report demonstrates that nonenzymatic glycation of Rubisco by
ascorbate results in increased susceptibility to proteases
and suggests that glycation could play a role in determining the in vivo turnover of Rubisco [161].
Conclusions
Perhaps no other enzyme so critical in the biology on
this planet is subject to such extensive processing and
constant conformational changes as Rubisco. In many
cases these modifications and structural changes are
essential for the functional role of Rubisco in photosynthetic CO2 assimilation, whereas for others the
functional role(s) remain unknown. Rubisco has served
science well as a model through which a number of
unique and widespread protein modifications and processes have been characterized. Although a great deal of
information is known about Rubisco, the constant and
frequent discovery of new information suggests that
many more important discoveries about the life of Rubisco remain to be elucidated.
Acknowledgment
The authors are very grateful to Dr. Robert J. Spreitzer for his assistance and editing during the preparation of this review.
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