Rubisco small-subunit α-helices control pyrenoid formation inChlamydomonas

Meyer, Moritz T.; Genkov, Todor; Skepper, Jeremy N.; Jouhet, Juliette; Mitchell, Madeline; Spreitzer, Robert J.; Griffiths, Howard

doi:10.1073/pnas.1210993109

Cited by 111 publications

(124 citation statements)

References 45 publications

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“…Various strategies have been deployed to enhance the kinetic properties of Rubisco, targeting both RbcL (Sharwood et al, 2008;Satagopan and Spreitzer, 2008) and RbcS (Karkehabadi et al, 2005;Genkov et al, 2010;Ishikawa et al, 2011). Although genetic engineering of hybrid Rubiscos partially imparted the desired kinetic properties of the foreign Rubiscos, the effects were not always beneficial because of trade-offs in kinetic efficiency Satagopan and Spreitzer, 2008;Ishikawa et al, 2011), impaired photosynthetic growth (Sharwood et al, 2008;Genkov et al, 2010), loss of chloroplast pyrenoid and CO 2 -concentrating mechanisms (Genkov et al, 2010;Meyer et al, 2012), and, more commonly, problems with Rubisco folding and assembly (Sharwood et al, 2008;Whitney et al, 2009;Genkov et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli

2016

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

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Section: Introductionmentioning

confidence: 99%

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli

2016

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“…C i in the form of bicarbonate (HCO 3 − ) is pumped into the chloroplast by active transport (8,9), where it is converted to CO 2 by the carbonic anhydrase (CA) CAH3, which is localized in the thylakoid lumen (10,11). This mechanism elevates the CO 2 concentration in the rubisco-containing compartment, the pyrenoid (12)(13)(14). A critical component of the CCM required at air levels of CO 2 is the stromal soluble protein complex CrLCIB-LCIC (limiting CO 2 -inducible B and C protein in C. reinhardtii) (15,16).…”

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confidence: 99%

Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases

Jin

Sun

Wunder

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Aquatic microalgae have evolved diverse CO 2 -concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO 2 -fixing enzyme rubisco. The limiting CO 2 -inducible B protein (LCIB) is known to be essential for CCM function in Chlamydomonas reinhardtii. To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs. Three of the six homologs are functional carbonic anhydrases (CAs). We determined the crystal structures of LCIB and limiting CO 2 -inducible C protein (LCIC) from C. reinhardtii and a CA-functional homolog from Phaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical β-CAs. Our results identify the LCIB family as a previously unidentified group of β-CAs, and provide a biochemical foundation for their function in the microalgal CCMs.LCIB | limiting-CO 2 inducible protein | CO 2 -concentrating mechanism | photosynthesis | carbonic anhydrases

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“…Two mechanisms for Rubisco accumulation in the pyrenoid have been proposed: (i) Rubisco holoenzymes could bind each other directly through hydrophobic residues (20), or (ii) a linker protein may link Rubisco holoenzymes together (18,20). The second model is based on analogy to the well-characterized prokaryotic carbon concentrating organelle, the β-carboxysome, where Rubisco aggregation is mediated by a linker protein consisting of repeats of a Significance Eukaryotic algae, which play a fundamental role in global CO 2 fixation, enhance the performance of the carbon-fixing enzyme Rubisco by placing it into an organelle called the pyrenoid.…”

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confidence: 99%

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

Mackinder

Meyer

Mettler‐Altmann

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO 2 -fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO 2 . Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO 2 . We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.pyrenoid | Rubisco | carbon fixation | Chlamydomonas reinhardtii | CO 2 -concentrating mechanism

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Rubisco small-subunit α-helices control pyrenoid formation inChlamydomonas

Cited by 111 publications

References 45 publications

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

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