scholarly journals The state of oligomerization of Rubisco controls the rate of synthesis of the Rubisco large subunit in Chlamydomonas reinhardtii

2021 ◽  
Author(s):  
Wojciech Wietrzynski ◽  
Eleonora Traverso ◽  
Francis-André Wollman ◽  
Katia Wostrikoff
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Large Subunit ◽  
Assembly Process ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Organisms ◽  
The Core ◽  
Key Enzyme ◽  
Assembly Intermediate ◽  
Life On Earth

Abstract Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is present in all photosynthetic organisms and is a key enzyme for photosynthesis-driven life on Earth. Its most prominent form is a hetero-oligomer in which small subunits (SSU) stabilize the core of the enzyme built from large subunits (LSU), yielding, after a chaperone-assisted multistep assembly process, an LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii and a combination of site-directed mutants to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU are associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis, in which LSU translation is controlled by its ability to assemble with the SSU, via the mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model whereby the last assembly intermediate, an LSU8-RAF1 complex, provides the platform for SSU binding to form the Rubisco enzyme, and when SSU is not available, converts to a key regulatory form that exerts negative feed-back on the initiation of LSU translation.

scholarly journals The state of oligomerization of Rubisco controls the rate of LSU translation in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Wojciech Wietrzynski ◽  
Eleonora Traverso ◽  
Francis-André Wollman ◽  
Katia Wostrikoff
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Large Subunit ◽  
Small Subunit ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Organisms ◽  
Initiation Of Translation ◽  
Key Enzyme ◽  
Assembly Intermediate ◽  
Life On Earth

AbstractRibulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is a key enzyme for photosynthesis-driven life on Earth. While present in all photosynthetic organisms, its most prominent form is a hetero-oligomer in which a Small Subunit (SSU) stabilizes the core of the enzyme built from Large Subunits (LSU), yielding, after a chaperone-assisted multistep assembly, a LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii, and a combination of site-directed mutants, to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU is associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis which places translation of its large subunit under the control of its ability to assemble with the small subunit, by a mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model where the last assembly intermediate, an octameric LSU8-RAF1 complex which delivers LSU to SSU to form the Rubisco enzyme, converts to a key regulator form able to exert a negative feed-back on the initiation of translation of LSU, when SSU is not available.

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

2008 ◽  
Vol 411 (2) ◽  
pp. 241-247 ◽  
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 ◽  
Bisphosphate Carboxylase ◽  
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.

scholarly journals Occurrence, phylogeny and evolution of ribulose-1,5-bisphosphate carboxylase/oxygenase genes in obligately chemolithoautotrophic sulfur-oxidizing bacteria of the genera Thiomicrospira and Thioalkalimicrobium

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2159-2169 ◽  
Author(s):  
Tatjana P. Tourova ◽  
Elizaveta M. Spiridonova ◽  
Ivan A. Berg ◽  
Boris B. Kuznetsov ◽  
Dimitry Yu. Sorokin
Keyword(s):  
16S Rrna ◽  
Large Subunit ◽  
Gene Encoding ◽  
Bisphosphate Carboxylase ◽  
Phylogenetic Cluster ◽  
New Family ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Sulfur Oxidizing ◽  
Phylogeny And Evolution

The occurrence of the different genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key enzyme of the Calvin–Benson–Bassham cycle of autotrophic CO2 fixation, was investigated in the members of the genus Thiomicrospira and the relative genus Thioalkalimicrobium, all obligately chemolithoautotrophic sulfur-oxidizing Gammaproteobacteria. The cbbL gene encoding the ‘green-like’ form I RubisCO large subunit was found in all analysed species, while the cbbM gene encoding form II RubisCO was present only in Thiomicrospira species. Furthermore, species belonging to the Thiomicrospira crunogena 16S rRNA-based phylogenetic cluster also possessed two genes of green-like form I RubisCO, cbbL-1 and cbbL-2. Both 16S-rRNA- and cbbL-based phylogenies of the Thiomicrospira–Thioalkalimicrobium–Hydrogenovibrio group were congruent, thus supporting its monophyletic origin. On the other hand, it also supports the necessity for taxonomy reorganization of this group into a new family with four genera.

The carbonic anhydrase gene families of Chlamydomonas reinhardtii

2005 ◽  
Vol 83 (7) ◽  
pp. 780-795 ◽  
Author(s):  
Mautusi Mitra ◽  
Catherine B Mason ◽  
Ying Xiao ◽  
Ruby A Ynalvez ◽  
Scott M Lato ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Chlamydomonas Reinhardtii ◽  
Gene Families ◽  
Carbonic Anhydrases ◽  
Photosynthetic Organisms ◽  
Surrounding Environment ◽  
Concentrating Mechanism ◽  
Chloroplast Stroma ◽  
Carbonic Anhydrase Gene

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3–. Aquatic photosynthetic organisms have evolved different forms of CO2-concentrating mechanisms to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CO2-concentrating mechanisms is the critical roles played by various specially localized extracellular and intracellular CAs. There are three evolutionarily unrelated CA families designated α-, β-, and γ-CA. In the green alga, Chlamydomonas reinhardtii Dangeard, eight CAs have now been identified, including three α-CAs and five β-CAs. In addition, C. reinhardtii has another CA-like gene, Glp1 that is similar to known γ-CAs. To characterize these different CA isoforms, some of the CA genes have been overexpressed to determine whether the proteins have CA activity and to generate antibodies for in vivo immunolocalization. The CA proteins Cah3, Cah6, and Cah8, and the γ-CA-like protein, Glp1, have been overexpressed. Cah3, Cah6, and Cah8 have CA activity, but Glp1 does not. At least two of these proteins, Cah3 and Cah6, are localized to the chloroplast. Using immunolocalization and sequence analyses, we have determined that Cah6 is located to the chloroplast stroma and confirmed that Cah3 is localized to the chloroplast thylakoid lumen. Activity assays show that Cah3 is 100 times more sensitive to sulfonamides than Cah6. We present a model on how these two chloroplast CAs might participate in the CO2-concentrating mechanism of C. reinhardtii. Key words: carbonic anhydrase, CO2-concentrating mechanism, Chlamydomonas, immunolocalization.

scholarly journals Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii

2002 ◽  
Vol 366 (3) ◽  
pp. 989-998 ◽  
Author(s):  
Cédric INVERNIZZI ◽  
Jonathan IMHOF ◽  
Gabriela BURKARD ◽  
Katharina SCHMID ◽  
Arminio BOSCHETTI
Keyword(s):  
Amino Acids ◽  
Chlamydomonas Reinhardtii ◽  
Small Subunit ◽  
Ribulose Bisphosphate Carboxylase ◽  
Cleavage Sites ◽  
Bisphosphate Carboxylase ◽  
Molecular Masses ◽  
Processing Site

The role of the two processing sites in the precursor of the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSS) of Chlamydomonas reinhardtii was studied by introducing mutations at the cleavage sites for the stromal processing peptidases SPP-1 and SPP-2, which hydrolyse wild-type pSS (20.6kDa) to an intermediate-sized product iSS (18.3kDa) and to the mature SS (16.3kDa), respectively. The mutations introduced into cDNA resulted in exchange of (a) two amino acids flanking processing site 1, or (b) one or (c) both amino acids flanking processing site 2. Mutation (a) prevented pSS from being processed at site 1 but not from cleavage at site 2. Mutation (c) abolished the action of SPP-2 but not SPP-1. When pSS with mutation (c) was imported into isolated chloroplasts, iSS accumulated while SS formation was abolished. However, mature SS was produced even in the absence of iSS synthesis (mutation a). Import of pSS bearing mutation (b), which only partially inhibited processing at the SPP-2 site, slowed the rate of SS formation down whereas iSS and some slightly smaller derivatives accumulated. These experiments suggested that in Chlamydomonas processing of pSS can occur in two steps, whereby the first step is facultative. The same three mutations were studied in vivo after transformation of SS-deficient C. reinhardtii T60-3 with mutated genomic DNA. Growth and photosynthesis was as in control transformants, except for the slower-growing transformants (mutation c) where no mature SS was immuno-detected. However, pSS fragments with molecular masses between those of iSS and SS were present even in the ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme.

scholarly journals Discovery of a readily heterologously expressed Rubisco from the deep sea with potential for CO2 capture

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Junli Zhang ◽  
Guoxia Liu ◽  
Alonso I. Carvajal ◽  
Robert H. Wilson ◽  
Zhen Cai ◽  
...  
Keyword(s):  
Deep Sea ◽  
Carbon Capture ◽  
Simple Structure ◽  
Large Subunit ◽  
Carboxylation Efficiency ◽  
Riftia Pachyptila ◽  
Bisphosphate Carboxylase ◽  
High Functioning ◽  
Highly Active

AbstractRibulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key CO2-fixing enzyme in photosynthesis, is notorious for its low carboxylation. We report a highly active and assembly-competent Form II Rubisco from the endosymbiont of a deep-sea tubeworm Riftia pachyptila (RPE Rubisco), which shows a 50.5% higher carboxylation efficiency than that of a high functioning Rubisco from Synechococcus sp. PCC7002 (7002 Rubisco). It is a simpler hexamer with three pairs of large subunit homodimers around a central threefold symmetry axis. Compared with 7002 Rubisco, it showed a 3.6-fold higher carbon capture efficiency in vivo using a designed CO2 capture model. The simple structure, high carboxylation efficiency, easy heterologous soluble expression/assembly make RPE Rubisco a ready-to-deploy enzyme for CO2 capture that does not require complex co-expression of chaperones. The chemosynthetic CO2 fixation machinery of chemolithoautotrophs, CO2-fixing endosymbionts, may be more efficient than previously realized with great potential for next-generation microbial CO2 sequestration platforms.

Protein synthesis in flax following inoculation with flax rust

1986 ◽  
Vol 64 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Ben C. S. Sutton ◽  
Michael Shaw
Keyword(s):  
Protein Synthesis ◽  
Large Subunit ◽  
Two Dimensional ◽  
Bisphosphate Carboxylase ◽  
Two Dimensional Electrophoresis ◽  
Host Pathogen Interaction ◽  
Melampsora Lini ◽  
In Vivo Labelling ◽  
Dimensional Electrophoresis

Resistance to flax rust Melampsora lini (Ehrenb.) Lév. in flax carrying the N resistance gene is determined by 24 h postinoculation, at which time hypersensitivity is observed. We have examined protein synthesis in cotyledons inoculated with both virulent and avirulent races of rust by in vivo labelling with [35S]methionine. The pattern of protein synthesis was assessed by one- and two-dimensional electrophoresis 8, 13, and 18 h after inoculation. No changes in protein synthesis were observed in the first 14 h following inoculation; however, by 18 h after inoculation the susceptible combination showed a marked decrease in protein synthesis (22%; P = 0.01). This could be largely accounted for by the reduced synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit, which was readily quantified on electrophoresis gels. In addition, a 30-kDa polypeptide also declined in the susceptible combination. Two-dimensional electrophoresis enabled changes to be detected in the synthesis of other minor polypeptides. None of these changes were observed in the resistant combination in which a small increase in the synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit and the 30-kDa polypeptide was found. These results indicate that the outcome of the host–pathogen interaction has already been determined by 18 h after inoculation.

scholarly journals Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons.

1985 ◽  
Vol 5 (9) ◽  
pp. 2238-2246 ◽  
Author(s):  
J O Berry ◽  
B J Nikolau ◽  
J P Carr ◽  
D F Klessig
Keyword(s):  
Specific Activity ◽  
Large Subunit ◽  
Growth Conditions ◽  
Small Subunit ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Bisphosphate Carboxylase

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

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