Reduced CO2/O2 specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas.

Chen, Zhixiang; Chastain, Chris J.; Al-Abed, Souhail R.; Chollet, Raymond; Spreitzer, Robert J.

doi:10.1073/pnas.85.13.4696

Cited by 89 publications

(100 citation statements)

References 32 publications

(59 reference statements)

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“…1B). We doubt that the large subunits are degraded as fast as they are synthesized during pulse labeling because a normal rate of large-subunit synthesis has been observed in an rbcL nonsense mutant (31,42), as well as in all rbcL missense mutants that have unstable holoenzymes (5,6,43). This raises the possibility that smallsubunit expression controls Rubisco subunit stoichiometry by regulating large-subunit expression at the level of translation.…”

Section: Fig 2 Organization Of the Rbcs Locus In C Reinhardtiimentioning

confidence: 91%

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Khrebtukova

Spreitzer

1996

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

108

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Ribulose-1,5-bisphosphate carboxylase͞ oxygenase (EC 4.1.1.39) is the key photosynthetic enzyme that catalyzes the first step of CO 2 fixation. The chloroplastlocalized holoenzyme of plants and green algae contains eight nuclear-encoded small subunits and eight chloroplastencoded large subunits. Although much has been learned about the enzyme active site that resides within each large subunit, it has been difficult to assess the role of eukaryotic small subunits in holoenzyme function and expression. Small subunits are coded by a family of genes, precluding genetic screening or nuclear transformation approaches for the recovery of small-subunit mutants. In this study, the two small-subunit genes of the green alga Chlamydomonas reinhardtii were eliminated during random insertional mutagenesis. The photosynthesis-deficient deletion mutant can be complemented with either of the two wild-type small-subunit genes or with a chimeric gene that contains features of both. Thus, either small subunit is sufficient for holoenzyme assembly and function. In the absence of small subunits, expression of chloroplast-encoded large subunits appears to be inhibited at the level of translation.

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Section: Fig 2 Organization Of the Rbcs Locus In C Reinhardtiimentioning

confidence: 91%

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Khrebtukova

Spreitzer

1996

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

108

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“…The kinetic constants of purified and activated Rubisco were measured by the incorporation of acid-stable 14 C from NaH 14 CO 3 (55). (56,58).…”

Section: Methodsmentioning

confidence: 99%

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits

Genkov

Meyer

Griffiths

et al. 2010

Journal of Biological Chemistry

138

105

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There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO 2 fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO 2 /O 2 specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO 2 /O 2 specificity but a lower carboxylation V max than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO 2 /O 2 specificity and retain near normal V max values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO 2 is concentrated for optimal photosynthesis.Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 2 EC 4.1.1.39) is the key photosynthetic enzyme responsible for CO 2 fixation. In plants and green algae, eight ϳ16-kDa nuclear encoded small subunits assemble with eight ϳ55-kDa large subunits in the chloroplast to form the functional holoenzyme (reviewed in Ref. 1). The chloroplast-encoded large subunit contains the ␣/␤-barrel active site at which CO 2 and O 2 compete for substrate ribulose-1,5-bisphosphate (RuBP) (reviewed in Refs. 1-3). Photosynthetic CO 2 fixation depends on the Rubisco V max of carboxylation (V c ) and the K m for CO 2 (K c ), but net CO 2 fixation is decreased by competitive inhibition from O 2 and the loss of CO 2 in photorespiration, which are defined by the V max of oxygenation (V o ) and the K m for O 2 (4). The ratio of the catalytic efficiencies of carboxylation (V c /K c ) to oxygenation (V o /K o ) defines the CO 2 /O 2 specificity factor (⍀) (4, 5). Because ⍀ is determined by the difference between the free energies of activation for carboxylation and oxygenation at the rate-determining step of catalysis (6), there is much interest in defining the structural basis for variation in this kinetic constant. The catalytic properties of Rubisco vary among divergent species (7-9), indicating that it might be possible to engineer improvements in Rubisco function (1). However, some plant and algal sp...

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“…One photosynthesis-competent revertant of a temperatureconditional rbcL mutant has been enigmatic (11)(12)(13). It results from a mutation in a nuclear gene, named Rbc-1, the product of which acts posttranslationally to correct decreases in holoenzyme stability and ⍀ caused by a Leu 290 -to-Phe (L290F) largesubunit substitution (13).…”

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confidence: 99%

RbcS suppressor mutations improve the thermal stability and CO ₂ /O ₂ specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase

Hong²,

Spreitzer³

2000

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

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In the green alga Chlamydomonas reinhardtii, a Leu 290 -to-Phe (L290F) substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), which is coded by the chloroplast rbcL gene, was previously found to be suppressed by second-site Ala 222 -to-Thr and Val 262 -to-Leu substitutions. These substitutions complement the photosynthesis deficiency of the L290F mutant by restoring the decreased thermal stability, catalytic efficiency, and CO 2͞O2 specificity of the mutant enzyme back to wild-type values. Because residues 222, 262, and 290 interact with the loop between ␤ strands A and B of the Rubisco small subunit, which is coded by RbcS1 and RbcS2 nuclear genes, it seemed possible that substitutions in this loop might also suppress L290F. A mutation in a nuclear gene, Rbc-1, was previously found to suppress the biochemical defects of the L290F enzyme at a posttranslational step, but the nature of this gene and its product remains unknown. In the present study, three nuclear-gene suppressors were found to be linked to each other but not to the Rbc-1 locus. DNA sequencing revealed that the RbcS2 genes of these suppressor strains have mutations that cause either Asn 54 -to-Ser or Ala 57 -to-Val substitutions in the small-subunit ␤A͞␤B loop. When present in otherwise wild-type cells, with or without the resident RbcS1 gene, the mutant small subunits improve the thermal stability of wild-type Rubisco. These results indicate that the ␤A͞␤B loop, which is unique to eukaryotic Rubisco, contributes to holoenzyme thermal stability, catalytic efficiency, and CO 2͞O2 specificity. The small subunit may be a fruitful target for engineering improved Rubisco. T he green alga Chlamydomonas reinhardtii serves as an excellent model for the study of photosynthesis because photosynthesis-deficient mutants can be maintained with acetate as an alternative source of carbon and energy. Mutations can be assigned to the nuclear or chloroplast genetic compartments by virtue of Mendelian or uniparental inheritance, and both compartments can be transformed with homologous or heterologous DNA (1). These attributes have been particularly useful for examining the structure-function relationships of eukaryotic ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco, Enzyme Commission 4.1.1.39) (2). Because land plants cannot be maintained in the complete absence of photosynthesis, and the subunits of eukaryotic Rubisco cannot be assembled in Escherichia coli (3), it has been difficult to examine the effects of random or directed mutations on the function of land-plant Rubisco in vivo or in vitro (4-6). Tobacco is the only land plant in which the chloroplast genome can be transformed (5, 6).By screening for photosynthesis-deficient Chlamydomonas mutants and selecting for photosynthesis-competent revertants, several regions of the Rubisco large subunit have been identified that influence the ratio of ribulose 1,5-bisphosphate (RuBP) carboxylase to oxygenase activities (2, 7). Because CO 2 and O 2 compete at the same la...

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Reduced CO2/O2 specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas.

Cited by 89 publications

References 32 publications

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits

RbcS suppressor mutations improve the thermal stability and CO ₂ /O ₂ specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase

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Reduced CO2/O2 specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas.

Cited by 89 publications

References 32 publications

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits

RbcS suppressor mutations improve the thermal stability and CO 2 /O 2 specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase

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RbcS suppressor mutations improve the thermal stability and CO ₂ /O ₂ specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase