Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Thermophilic Red Algae with a Strong Specificity for CO2Fixation

Uemura, Kenichiro; Anwaruzzaman,; Miyachi, Shigetoh; Yokota, Akiho

doi:10.1006/bbrc.1997.6497

Cited by 165 publications

(124 citation statements)

References 20 publications

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“…Published data for the influence of temperature on the specificities of the Rubiscos of spinach (32) and the red alga, Galdieria partita (33), clearly exemplify this phenomenon. Although the algal enzyme has a S c/o value more than twice that of the spinach protein at 25°C, the Arrhenius-style plots cross at 57°C (Fig.…”

Section: Possible Compensation Mechanismsmentioning

confidence: 84%

“…The higher the CO2͞O2 specificity, the more it is eroded by increasing temperature. (A) Arrhenius-style plots of the specificities of the Rubiscos from spinach (F) (32) and the red alga, Galdieria partita (■) (33 …”

Section: Discussionmentioning

confidence: 99%

“…Inferred from the carbon-isotope fractionation at saturating CO2 of whole tissues of another temperate floridiophycean macrophyte, Lamanea mamillosa (58). **For Rubisco isolated from the closely related species Galdieria partita (33).…”

Section: Possible Compensation Mechanismsmentioning

confidence: 99%

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Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

Tcherkez

Farquhar

Andrews

2006

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

678

709

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The cornerstone of autotrophy, the CO2-fixing enzyme, D-ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO2 and O2 as substrates, an ''abominably perplexing'' puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO2 molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO2͞O2 specificity, in response to reducing atmospheric CO2:O2 ratios, has resulted in a transition state for CO2 addition in which the CO2 moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO2, O2, and thermal conditions in their subcellular environments, optimizing this compromise between CO2͞O2 specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO2͞O2 specificity and 12 C͞ 13 C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature. enzyme mechanisms ͉ isotope fractionation ͉ transition states T he most abundant protein in nature is D-ribulose 1,5-bisphosphate (RuBP) carboxylase͞oxygenase (Rubisco, EC 4.1.1.39) (1). This immense N investment is required to counter the enzyme's pitifully sluggish catalytic performance. Furthermore, Rubisco's tendency to confuse the substrate of photosynthesis, CO 2 , with the product, O 2 , saddles all aerobic photosynthetic organisms with energy-wasting photorespiration (2). Thus, this single enzyme's efficiency, or lack thereof, dictates the (in)efficiency with which plants use their basic resources of light, water, and N, and currently intense biotechnological effort aims to improve its catalytic properties and to engineer such improvements into crop plants (3, 4). Rubisco's difficulties stem from the inevitable O 2 sensitivity of the 2,3-enediol form of RuBP, to which CO 2 is added during the carboxylase reaction (5), which causes carboxylase͞oxygenase bifunctionality (Fig. 1). This difficulty is exacerbated by the need to discriminate between featureless molecules, CO 2 and O 2 , that can be bound in Michaelis-Menten complexes only weakly, if at all (2, 6). R...

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Section: Possible Compensation Mechanismsmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

Tcherkez

Farquhar

Andrews

2006

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

678

709

View full text Add to dashboard Cite

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“…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)(8)(9), indicating that it might be possible to engineer improvements in Rubisco function (1). However, some plant and algal species contain CO 2 -concentrating mechanisms (CCMs) (reviewed in Refs.…”

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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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“…We have carried out the mutation experiments on loop 6 of Tk-Rubisco, which harbors the active site Lys-322 interacting with the incoming gaseous substrate (27,28), and the adjacent ␣-helix 6 (29). Replacement of the entire ␣-helix 6 of Tk-Rubisco with the corresponding region of the enzyme from spinach (SpRubisco), whose activity is higher than that of Tk-Rubisco at ambient temperatures (30), resulted in a 30% increase in turnover number for the carboxylase activity at 25°C in vitro (SP6 mutant; Fig. 1A) (29).…”

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confidence: 99%

Structure-based Catalytic Optimization of a Type III Rubisco from a Hyperthermophile

Nishitani

Yoshida

Fujihashi

et al. 2010

Journal of Biological Chemistry

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The Calvin-Benson-Bassham cycle is responsible for carbon dioxide fixation in all plants, algae, and cyanobacteria. The enzyme that catalyzes the carbon dioxide-fixing reaction is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) belongs to the type III group, and shows high activity at high temperatures. We have previously found that replacement of the entire ␣-helix 6 of Tk-Rubisco with the corresponding region of the spinach enzyme (SP6 mutant) results in an improvement of catalytic performance at mesophilic temperatures, both in vivo and in vitro, whereas the former and latter half-replacements of the ␣-helix 6 (SP4 and SP5 mutants) do not yield such improvement. We report here the crystal structures of the wild-type Tk-Rubisco and the mutants SP4 and SP6, and discuss the relationships between their structures and enzymatic activities. A comparison among these structures shows the movement and the increase of temperature factors of ␣-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the ␣-helix 6 and loop 6 regions was important to increase the catalytic activity of Tk-Rubisco at ambient temperatures. Based on this structural information, we constructed a new mutant, SP5-V330T, which was designed to have significantly greater flexibility in the above region, and it proved to exhibit the highest activity among all mutants examined to date. The thermostability of the SP5-V330T mutant was lower than that of wild-type Tk-Rubisco, providing further support on the relationship between flexibility and activity at ambient temperatures.The Calvin-Benson-Bassham (CBB) 4 cycle contributes to the fixation of carbon for all plants, algae, and cyanobacteria. The most important enzyme in the CBB cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco: EC 4.1.1.39) (1, 2). Rubisco is responsible for the single carbon dioxidefixing step of the cycle, in which ribulose-1,5-bisphosphate reacts with CO 2 and H 2 O to form two molecules of 3-phosphoglycerate. However, it also catalyzes a competitive oxygenase reaction, by which ribulose-1,5-bisphosphate reacts with O 2 to form one molecule of 3-phosphoglycerate and one molecule of 2-phosphoglycolate (3, 4). Moreover, a typical Rubisco is able to fix only three carbon dioxide molecules/s, an extremely low turnover rate for an enzyme (3). Rubisco is thus considered to be the rate-limiting enzyme of the CBB pathway, and has been an important target for protein engineering (3).From sequence alignment, Rubiscos can be classified into four groups, types I, II, III, and IV (5). The classical type I and II Rubiscos function in the CBB cycle. Type I Rubisco is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L 8 S 8 ) (6). Type II Rubisco is usually composed of only two L subunits (L 2 ) (7, 8). In both cases, the L 2 dimer is the catalytic unit of Rubisco, generating two catalytic...

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Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Thermophilic Red Algae with a Strong Specificity for CO2Fixation

Cited by 165 publications

References 20 publications

Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

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

Structure-based Catalytic Optimization of a Type III Rubisco from a Hyperthermophile

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