Activase Region on Chloroplast Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

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Rubisco activase is an AAA؉ protein, a superfamily with members that use a "Sensor 2" domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases were constructed, interchanging a Sensor 2-containing region between activases from spinach and tobacco. Spinach chimeric activase was a poor activator of both spinach and tobacco Rubisco. In contrast, tobacco chimeric activase activated spinach Rubisco far better than tobacco Rubisco, similar to spinach activase. A point mutation, K311D, in the Sensor 2 domain of the tobacco chimeric activase abolished its ability to better activate spinach Rubisco. The opposite mutation, D311K, in wild type tobacco activase produced an enzyme that activated both spinach and tobacco Rubisco, whereas a second mutation, D311K/L314V, shifted the activation preference toward spinach Rubisco. The involvement of these two residues in substrate selectivity was confirmed by introducing the analogous single and double mutations in cotton activase. The ability of the two tobacco activase mutants to activate wild type and mutant Chlamydomonas Rubiscos was also examined. Tobacco D311K activase readily activated wild type and P89R but not D94K Rubisco, whereas the tobacco L314V activase only activated D94K Rubisco. The tobacco activase double mutant D311K/L314V activated wild type Chlamydomonas Rubisco better than either the P89R or D94K Rubisco mutants, mimicking activation by spinach activase. The results identified a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco.Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) 1 activase is a chloroplast protein that activates and maintains Rubisco in an active state by facilitating removal of various sugar phosphates that either block substrate binding or prevent carbamoylation (1, 2). Plants lacking activase or having a very low level of activase cannot survive at atmospheric CO 2 levels (3, 4), and those expressing reduced levels exhibit reduced rates of photosynthesis and growth (5, 6). The activation process requires ATP hydrolysis (7), but this activity, as measured in vitro, appears to be related to self-association, which occurs even in the absence of Rubisco (8). The activase is subjected to redox regulation in Arabidopsis and probably other plant genera (9, 10). The proposed mechanism of action includes a binding step between activase and Rubisco, which is consistent with the kinetics of the activation process (11, 12).Although chemical cross-linking (13) and co-immunoprecipitation of Rubisco and activase have been reported (14, 15), most of the details of the binding process are still largely unknown. However, some insight into the process has been obtained by exploiting the peculiar specificity of Rubisco activase from plants in the family Solanaceae. Activase from plants in this family, which includes tobacco, tomato, pota...

mentioning

confidence: 50%

Section: Activation Of Chlamydomonas Rubiscos By Tobacco Activasementioning

confidence: 99%

Two Residues of Rubisco Activase Involved in Recognition of the Rubisco Substrate

Salvucci

Portis

2005

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“…One might consider that interactions between Rubisco and Rubisco activase have been altered in the hybrid enzyme mutants (1). However, a mutant of Chlamydomonas that lacks a functional activase gene grows as well as wild type at air levels of CO 2 (71), and the species specificity of Rubisco activase interactions has been shown to occur via the Rubisco large subunit (72,73).…”

Section: Resultsmentioning

confidence: 99%

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

Genkov

Meyer

Griffiths

et al. 2010

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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...

“…RuBP carboxylase and oxygenase activities were measured via the incorporation of acid-stable 14 C from NaH 14 CO 3 (45). The ratio of carboxylation to oxygenation at any given concentrations of CO 2 .…”

Section: Strains and Culture Conditions-c Reinhardtii 2137 Mtmentioning

confidence: 99%

“…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 of a neighboring large subunit (reviewed in Ref. 4), which also contributes residues to the active site and may contain the primary site of interaction with Rubisco activase (13,14). In the absence of RuBP or CABP, the carboxyl terminus is disordered, and loop 6 is usually disordered or misfolded into an open conformation depending on the source or treatment of the Rubisco used for crystallization (15)(16)(17)(18)(19)).…”

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 ...