Rubisco activase remodels plant Rubisco via the large subunit N-terminus

Interaction Sites ◽

Highly Sensitive ◽

N Terminus ◽

Sugar Phosphates

ABSTRACTThe photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms inhibited complexes with multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. At least three classes of ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas) have evolved to remodel inhibited Rubisco complexes. The mechanism of green-type Rca found in higher plants has proved elusive, because until recently higher plant Rubiscos could not be expressed recombinantly. Towards identifying interaction sites between Rubisco and Rca, here we produce and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. We find that Rca activity is highly sensitive to truncations and mutations in the conserved N-terminus of the Rubisco large subunit. Both T5A and T7A substitutions cannot be activated by Rca, but present with increased carboxylation velocities. Our results are consistent with a model where Rca functions by transiently threading the Rubisco large subunit N-terminus through the axial pore of the AAA+ hexamer.

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015759 ◽

2020 ◽

Vol 295 (48) ◽

pp. 16427-16435

Author(s):

Jediael Ng ◽

Zhijun Guo ◽

Oliver Mueller-Cajar

Keyword(s):

Higher Plants ◽

Large Subunit ◽

Rubisco Activase ◽

Higher Plant ◽

Interaction Sites ◽

Dead End ◽

Highly Sensitive ◽

N Terminus ◽

Sugar Phosphates

The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1–4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer.

Probing the rice Rubisco–Rubisco activase interaction via subunit heterooligomerization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914245116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24041-24048 ◽

Cited By ~ 10

Author(s):

Devendra Shivhare ◽

Jediael Ng ◽

Yi-Chin Candace Tsai ◽

Oliver Mueller-Cajar

Keyword(s):

Active Sites ◽

Crop Improvement ◽

Fold Increase ◽

Rubisco Activase ◽

Growth And Yield ◽

Higher Plant ◽

Functional Understanding ◽

Sugar Phosphates ◽

Aaa Protein

During photosynthesis the AAA+ protein and essential molecular chaperone Rubisco activase (Rca) constantly remodels inhibited active sites of the CO2-fixing enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) to release tightly bound sugar phosphates. Higher plant Rca is a crop improvement target, but its mechanism remains poorly understood. Here we used structure-guided mutagenesis to probe the Rubisco-interacting surface of rice Rca. Mutations in Ser-23, Lys-148, and Arg-321 uncoupled adenosine triphosphatase and Rca activity, implicating them in the Rubisco interaction. Mutant doping experiments were used to evaluate a suite of known Rubisco-interacting residues for relative importance in the context of the functional hexamer. Hexamers containing some subunits that lack the Rubisco-interacting N-terminal domain displayed a ∼2-fold increase in Rca function. Overall Rubisco-interacting residues located toward the rim of the hexamer were found to be less critical to Rca function than those positioned toward the axial pore. Rca is a key regulator of the rate-limiting CO2-fixing reactions of photosynthesis. A detailed functional understanding will assist the ongoing endeavors to enhance crop CO2 assimilation rate, growth, and yield.

Immunolocalization of Rubisco Activase and Rubisco in C3 and C4 Plant Tissues

Microscopy and Microanalysis ◽

10.1017/s1431927600034851 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 472-473

Author(s):

S. Madhavan ◽

M. S. Miller-Goodman ◽

K. W. Lee

Keyword(s):

Higher Plants ◽

Tight Binding ◽

Rubisco Activase ◽

Ribulose Bisphosphate ◽

Ribulose Bisphosphate Carboxylase Oxygenase ◽

Microscopic Studies ◽

Sugar Phosphates

Ribulose bisphosphate carboxylase/oxygenase (Rubisco), an abundant enzyme in chloroplasts, must be activated by CO2 in order for it to catalyze the carboxylation of ribulose bisphosphate. Rubisco activase, a nuclear encoded chloroplast protein was first identified as a biochemical lesion in the rca mutant of Arabidopsis (1) which lacked this enzyme. Study of Rubisco in this mutant (2) and transgenic tobacco plants with reduced Rubisco activase levels showed that Rubisco could not achieve and maintain an adequate level of activity, in vivo, without an activase. Rubisco activase promotes ‘activation’ of Rubisco by overcoming the deleterious effects of tight binding sugar phosphates and low chloroplast CO2 levels on catalysis and carbamylation (1).Rubisco activase has been detected in higher plants (3), in unicellular green algae (4,5) and in cyanobacteria (6). Though the presence of Rubisco in guard cell chlroplasts was a subject of controversy, several immunolight and immunoelectron microscopic studies have demonstrated the presence of Rubisco in guard cells (7).

Limitation of the Rate of Ribulosebisphosphate Carboxylase Activation by Carbamylation and Ribulosebisphosphate Carboxylase Activase Activity: Development and Tests of a Mechanistic Model

Functional Plant Biology ◽

10.1071/pp9960141 ◽

1996 ◽

Vol 23 (2) ◽

pp. 141 ◽

Cited By ~ 20

Author(s):

IE Woodrow ◽

ME Kelly ◽

KA Mott

Keyword(s):

Mechanistic Model ◽

Large Subunit ◽

Intercellular Co2 Concentration ◽

Rubisco Activase ◽

Photon Flux ◽

Photon Flux Density ◽

Ribulosebisphosphate Carboxylase ◽

Kinetics Of ◽

Rubisco Activation

A mechanistically-based model of light-mediated activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is developed. The model describes the kinetics of Rubisco activation following a relatively rapid increase in photon flux density (PPFD) from an initially low level. Underlying the model is the assumption that there are two slow processes that could potentially limit the rate of light-mediated Rubisco activation. These processes are the addition of the activator CO2 to the large subunit of Rubisco, which is accompanied by a conformational change in the enzyme (carbamylation), and activase-mediated removal of ribulose 1,5-bisphosphate from the inactive form of the enzyme. The contribution of these slow processes to the overall activation kinetics of Rubisco was resolved by measuring Rubisco activation in whole spinach leaves using non-steady-state CO2 exchange. It was found that when the change in PPFD was relatively small and a correspondingly small proportion of the Rubisco pool was activated, the kinetics of activation were highly sensitive to the intercellular CO2 concentration (ci). The apparent rate constant for activation under these conditions was found to be similar to that for the carbamylation of purified spinach Rubisco. When the change in PPFD and the proportion of Rubisco activated was relatively large, however, the kinetics of Rubisco activation were almost completely CO2 insensitive and were consistent with those of an enzyme-catalysed reaction. It is suggested that (1) CO2-insensitive activation reflects the operation of Rubisco activase and (2) the increasing CO2 sensitivity seen as the change in PPFD decreases reflects a transition to limitation by carbamylation.

The study of plant phylogeny using amino acid sequences of Ribulose-1,5-Bisphosphate carboxylase. I. Patterns of variability

Australian Journal of Botany ◽

10.1071/bt9830395 ◽

1983 ◽

Vol 31 (4) ◽

pp. 395 ◽

Cited By ~ 8

Author(s):

PG Martin ◽

AC Jennings

Keyword(s):

Amino Acids ◽

Large Subunit ◽

Small Subunit ◽

Amino Acid Sequences ◽

Protein Sequencing ◽

Plant Phylogeny ◽

Variable Site ◽

N Terminus

Ribulose bisphosphate carboxylase has been prepared from 50 species of angiosperms from 16 diverse families. In 35 preparations, well known 'bland leaf' methods were used but 15 species had 'pungent leaves' and for these a new preparative method is described. Automatic methods have been used to obtain N-terminal sequences (40 amino acids) of the small subunit (SSU) from all 50 species and the pattern of variability is discussed: 26 of 40 positions are variable to a degree similar to that found in plastocyanin and plant cytochrome c, i.e, an average of 3.7 different amino acids per variable site. These results, and the fact that sufficient protein can be obtained from 100 g of leaves, make a widespread phylogenetic survey of angiosperm SSU feasible and it is claimed that the method is at least as practicable as nucleic acid sequencing. A limited amount of sequencing has been carried out on the large subunit (LSU) but its low variability discourages a protein sequencing survey. Implications for gene structure and function are discussed and evidence is given that active LSU is derived from a precursor with 14 additional amino acids at the N-terminus. In SSU, variability of the two N- terminal amino acids suggests that they are not involved in the signals for removal of either the transit peptide or, in the RNA, of the intron, excision of one end of which depends on the codons for the invariable amino acids at positions 3 and 4. Evidence is also given that if the N-terminus of SSU is methionine, as is common, then it is modified and associated with a 'frayed' N-terminus.

Photoregulation of the biosynthesis of ribulose bisphosphate carboxylase

Development ◽

10.1242/dev.83.supplement.163 ◽

1984 ◽

Vol 83 (Supplement) ◽

pp. 163-178

Author(s):

R. John Ellis ◽

Thomas F. Gallagher ◽

Gareth I. Jenkins ◽

C. Ruth Lennox

Keyword(s):

Chloroplast Development ◽

Higher Plants ◽

Large Subunit ◽

Nuclear Genome ◽

Small Subunit ◽

Ribulose Bisphosphate ◽

Subunit Mrna ◽

Parallel Increase

Chloroplast development in higher plants is light dependent, and is accompanied by the synthesis of chlorophyll and the accumulation of many chloroplast polypeptides. There is a 100-fold greater content of the photosynthetic enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase, in light-grown seedlings of Pisum sativum than in dark-grown seedlings. Following the illumination of dark-grown seedlings, there is a parallel increase in the content of both the mRNA and the polypeptide of the small subunit of the carboxylase; this subunit is a product of the nuclear genome. The increases in the mRNA and the polypeptide of the large subunit, which is a product of the chloroplast genome, show less synchronicity. Studies with isolated leaf nuclei show that the increase in small subunit mRNA is mediated primarily at the level of transcription. Three distinct effects of light on transcription of small subunit genes have been found; a rapid (∼1 h) burst, followed by a decline, when etiolated plants are first exposed to light; a slow (∼36h) development of the competence to transcribe rapidly after the initial burst; rapid (∼20 min) switches in both directions when fully greened plants are exposed to light—dark transitions.

Structure ofArabidopsis thalianaRubisco activase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715001182 ◽

2015 ◽

Vol 71 (4) ◽

pp. 800-808 ◽

Cited By ~ 23

Author(s):

Dirk Hasse ◽

Anna M. Larsson ◽

Inger Andersson

Keyword(s):

Conformational Changes ◽

Rubisco Activase ◽

Sulfate Ion ◽

Sulfate Ions ◽

Dead End ◽

Inhibitory Compounds ◽

Density Map ◽

Sugar Phosphates ◽

Electron Density Map

The CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inactivated by the formation of dead-end complexes with inhibitory sugar phosphates. In plants and green algae, the ATP-dependent motor protein Rubisco activase restores catalytic competence by facilitating conformational changes in Rubisco that promote the release of the inhibitory compounds from the active site. Here, the crystal structure of Rubisco activase fromArabidopsis thalianais presented at 2.9 Å resolution. The structure reveals an AAA+ two-domain structure. More than 100 residues in the protein were not visible in the electron-density map owing to conformational disorder, but were verified to be present in the crystal by mass spectrometry. Two sulfate ions were found in the structure. One was bound in the loop formed by the Walker A motif at the interface of the domains. A second sulfate ion was bound at the N-terminal end of the first helix of the C-terminal domain. The protein packs in a helical fashion in the crystal, as observed previously for Rubisco activase, but differences in the helical pitch indicate flexibility in the packing of the protein.

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein.

The Journal of Cell Biology ◽

10.1083/jcb.103.4.1327 ◽

1986 ◽

Vol 103 (4) ◽

pp. 1327-1335 ◽

Cited By ~ 43

Author(s):

S Cannon ◽

P Wang ◽

H Roy

Keyword(s):

Higher Plants ◽

Binding Protein ◽

Large Subunit ◽

Ribulose Bisphosphate ◽

Dual Effect ◽

Low Concentrations ◽

The One

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.

Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts

10.1101/2020.10.26.354332 ◽

2020 ◽

Author(s):

Nicky Atkinson ◽

Yuwei Mao ◽

Kher Xing Chan ◽

Alistair J. McCormick

Keyword(s):

Higher Plants ◽

Initial Step ◽

Small Subunit ◽

Operating Efficiency ◽

Higher Plant ◽

Spontaneous Condensation ◽

Concentrating Mechanism ◽

Chloroplast Stroma ◽

Almost All

SummaryPhotosynthetic CO2 fixation in plants is limited by the inefficiency of the CO2-assimilating enzyme Rubisco (D-ribulose-1,5-bisphosphate carboxylase/ oxygenase)1–3. In plants possessing the C3 pathway, which includes most major staple crops, Rubisco is typically evenly distributed throughout the chloroplast stroma. However, in almost all eukaryotic algae Rubisco aggregates within a microcompartment known as the pyrenoid, in association with a CO2-concentrating mechanism that improves photosynthetic operating efficiency under conditions of low inorganic carbon4. Recent work has shown that the pyrenoid matrix is a phase-separated, liquid-like condensate5. In the alga Chlamydomonas reinhardtii, condensation is mediated by two components: Rubisco and the linker protein EPYC1 (Essential Pyrenoid Component 1)6,7. Here we show that expression of mature EPYC1 and a plant-algal hybrid Rubisco leads to spontaneous condensation of Rubisco into a single phase-separated compartment in Arabidopsis chloroplasts, with liquid-like properties similar to a pyrenoid matrix. The condensate displaces the thylakoid membranes and is enriched in hybrid Rubisco containing the algal Rubisco small subunit required for phase separation. Promisingly, photosynthetic CO2 fixation and growth is not impaired in stable transformants compared to azygous segregants. These observations represent a significant initial step towards enhancing photosynthesis in higher plants by introducing an algal CO2-concentrating mechanism, which is predicted to significantly increase the efficiency of photosynthetic CO2 uptake8,9.

Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Biochemical Journal ◽

10.1042/bj20071422 ◽

2008 ◽

Vol 411 (2) ◽

pp. 241-247 ◽

Cited By ~ 8

Author(s):

María-Jesús García-Murria ◽

Saeid Karkehabadi ◽

Julia Marín-Navarro ◽

Sriram Satagopan ◽

Inger Andersson ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Higher Plants ◽

Large Subunit ◽

Dimensional Structure ◽

Wild Type ◽

Wild Type Enzyme ◽

Specificity Factor

Proximal Cys172 and Cys192 in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys172 and Cys192 by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The Vc (Vmax for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the Km for CO2 and O2 was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.