Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase:  Substitution at Arg-71 Affects Thermal Stability and CO<sub>2</sub>/O<sub>2</sub> Specificity

Spreitzer, Robert J.; Esquível, M. G.; Du, Yong; McLaughlin, Patrick D.

doi:10.1021/bi002943e

Cited by 40 publications

(61 citation statements)

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“…The rbcS1 gene is routinely used for experiments solely because it was observed to give a higher frequency of transformation than rbcS2 (35). Although Chlamydomonas cells containing only one rbcS gene have about half as much Rubisco as wild-type cells (35), Rubisco enzymes containing either small subunit 1 or small subunit 2 do not differ with respect to level of expression, catalytic properties, or thermal stability (19,20,35). Only one rbcS sequence is known for sunflower (32,46).…”

Section: Resultsmentioning

confidence: 99%

“…To determine whether the hybrid Rubisco enzymes may have an associated structural instability, thermal inactivation experiments were performed (19,54). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The cell extract was subjected to SDS-PAGE and Western blotting (51, 52) or fractionated on 10 -30% sucrose gradients prepared in the same extraction buffer (without polyvinylpyrrolidone) to isolate pure Rubisco holoenzyme (53). Thermal stability of purified Rubisco was determined as described previously (19,54).…”

Section: Methodsmentioning

confidence: 99%

“…16). Directed mutagenesis of Rubisco from the cyanobacterium Synechococcus expressed in Escherichia coli and directed mutagenesis and genetic selection in vivo in the green alga Chlamydomonas reinhardtii have identified several residues of the small subunit that can influence ⍀ (17)(18)(19)(20)(21). When the longer loop between ␤-strands A and B of the Chlamydomonas small subunit was replaced with the shorter loop of Synechococcus, a decrease in ⍀ was observed (15,22).…”

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

Genkov

Meyer

Griffiths

et al. 2010

Journal of Biological Chemistry

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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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Section: Resultsmentioning

confidence: 99%

“…To determine whether the hybrid Rubisco enzymes may have an associated structural instability, thermal inactivation experiments were performed (19,54). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Genkov

Meyer

Griffiths

et al. 2010

Journal of Biological Chemistry

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138

105

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“…4), it is a daunting task to identify regions of structure far from the active site that may influence catalysis. Only by genetic screening and selection in vivo (29), random or scanning mutagenesis (53)(54)(55), bioinformatic analysis of sequence divergence (56), and/or detailed comparative analysis of divergent x-ray crystal structures (18) may it be possible to identify regions worthy of experimental analysis. Based on an extensive comparison of Rubisco crystal structures, Duff et al (18) proposed that Asp-473 in the carboxyl-terminal region of the large subunit may serve as a critical latch residue that accounts for the folding of the carboxyl terminus over loop 6 during catalysis.…”

Section: Discussionmentioning

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

Journal of Biological Chemistry

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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 ...

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Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production

Pinto¹,

Malcata

Arrabaça

et al. 2013

Appl Microbiol Biotechnol

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Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48 h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10-15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae.

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Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity

Cited by 40 publications

References 37 publications

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

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

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

Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production

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Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO2/O2 Specificity

Cited by 40 publications

References 37 publications

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

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

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

Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production

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Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity