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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138

105

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...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Genkov

Meyer

Griffiths

et al. 2010

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138

105

“…The location of Arg-59 from a second S subunit in the Chlamydomonas structure is in gold. N54S and A57V S subunit substitutions (Chlamydomonas residues in yellow) complement a mutant Phe substitution at L subunit Leu-290 (in light blue) (38). Residues in a neighboring L subunit (dark blue) differ between Chlamydomonas (methylCys-256) and Spinacia (Phe-256).…”

Section: Figmentioning

confidence: 99%

First Crystal Structure of Rubisco from a Green Alga,Chlamydomonas reinhardtii

Taylor

Backlund

Björhall

et al. 2001

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125

The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 Å resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between ␤ strands A and B of the small subunit (␤A-␤B loop), which is longer by six amino acid residues than the corresponding region in Rubisco from Spinacia. Mutations of residues in the ␤A-␤B loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonas structure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.Ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39; Rubisco) 1 catalyzes the addition of CO 2 to RuBP (reviewed in Refs. 1-3). This reaction initiates photosynthetic carbon assimilation via the Calvin cycle and results in the net gain of carbon from atmospheric CO 2 into the biosphere. The carboxylation reaction of Rubisco is the major source of carbon needed for life. A competing reaction in which O 2 is added to RuBP results ultimately in a net loss of CO 2 , a process known as photorespiration. Because the reaction catalyzed by Rubisco is unique to photosynthetic CO 2 fixation and nearly all carbohydrate production is dependent on it, the oxygenase reaction qualifies as the most important limiting factor of photosynthetic yield.All Rubisco enzymes have oxygenase activity. This is true also for strictly anaerobic microbes, including some anaerobic archaebacteria (reviewed in Ref. 1). Thus, whereas the ratio of carboxylation to oxygenation at any specified concentrations of CO 2 and O 2 depends on the catalytic efficiency (k cat /K m ) of carboxylation relative to that of oxygenation, referred to as the CO 2 /O 2 specificity factor, net CO 2 fixation is determined by the difference between the rates of carboxylation and oxygenation (4, 5). The kinetic constants that determine carboxylation and oxygenation vary considerably among Rubisco enzymes from different species (6).Based on their quaternary structures, different forms of Rubisco enzymes can be distinguished (reviewed in Refs. 1 and 2). In cyanobacteria and plants with chloroplasts, such as red and brown algae and green plants, a hexadecameric (L8S8) form is found, consisting of eight 55-kDa L subunits and eight 15-kDa S subunits. A dimeric (L2) enzyme, similar in structure to the L subunits of the hexadecameric enzymes, is restricted to some bacteria and alveolates. More recently, a third form was discovered in the thermophilic archaebacterium Thermococcus kodakaraensis that comprises 10 L sub...

“…30). Furthermore, because mutant genes exist in vivo, genetic selection can be used to recover second-site suppressor mutations as a means for identifying complementing structural interactions (29,31). With this in mind, and considering the evolutionary conservation and potential critical function of Asp-473, we decided to examine the essentiality of Asp-473 by directed mutagenesis and Chlamydomonas chloroplast transformation.…”

mentioning

confidence: 99%

Substitutions at the Asp-473 Latch Residue of Chlamydomonas Ribulosebisphosphate Carboxylase/Oxygenase Cause Decreases in Carboxylation Efficiency and CO2/O2 Specificity

Satagopan

Spreitzer

2004

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The loop between ␣-helix 6 and ␤-strand 6 in the ␣/␤-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO 2 and O 2 . Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V max /K m ) and ϳ16% decreases in CO 2 /O 2 specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO 2 /O 2 specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco.Like many ␣/␤-barrel-domain enzymes (1), the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 1 EC 4.1.1.30) has a loop (i.e. between ␤-strand 6 and ␣-helix 6) that folds over substrate during catalysis (reviewed in Refs. 2-4). Numerous studies have indicated that loop 6 plays a major role in discriminating between CO 2 and O 2 in the competing RuBP carboxylation and oxygenation reactions of Rubisco (reviewed in Refs. 2 and 3), and Lys-334 at the apex of loop 6 interacts with the C-2 carboxyl group of the transition state analog CABP in various Rubisco x-ray crystal structures (5-9). However, Rubisco may be unique among ␣/␤-barrel enzymes in that the carboxyl terminus folds over loop 6 and appears to stabilize its closed conformation. This arrangement of loop 6 and the carboxyl terminus is also observed in the crystal structure of unactivated Rubisco that contains RuBP in the active site (10). In vivo, Rubisco activase is responsible for facilitating the opening of the closed structure to release this RuBP (reviewed in Refs. 2 and 11), thereby allowing spontaneous carbamylation of an active site Lys-201 and introduction of Mg 2ϩ to produce the active form of the enzyme (reviewed in Ref. 12). The change from closed to open conformation in the carboxyl-terminal, ␣/␤-barrel domain of the large subunit is accompanied by movement of the amino-terminal domain ...