Plant-like substitutions in the large-subunit carboxy terminus of Chlamydomonas Rubisco increase CO2/O2 Specificity

<p>Abstract</p> <p>Background</p> <p>Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from <it>Chlamydomonas reinhardtii </it>is ~90% identical to the flowering-plant sequences, although they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the <it>Chlamydomonas </it>large subunit to mimic the sequence of higher-specificity plant enzymes.</p> <p>Results</p> <p>The quadruple-mutant enzyme has a 10% increase in CO₂/O₂specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to influence the protein expression, structural stability or the function in vivo.</p> <p>Conclusion</p> <p>Owing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a "better" enzyme. Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches.</p

Plant-like Substitutions in the Large-Subunit Carboxy Terminus of \u3ci\u3eChlamydomonas\u3c/i\u3e Rubisco Increase CO\u3csub\u3e2\u3c/sub\u3e/O\u3csub\u3e2\u3c/sub\u3e Specificity

Background: Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is ~90% identical to the flowering-plant sequences, although they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the Chlamydomonas large subunit to mimic the sequence of higher-specificity plant enzymes. Results: The quadruple-mutant enzyme has a 10% increase in CO2/O2 specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to influence the protein expression, structural stability or the function in vivo. Conclusion: Owing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a better enzyme. Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches

Phylogenetic engineering of ribulose -1,5 -bisphosphate carboxylase /oxygenase large-subunit loop 6 in Chlamydomonas reinhardtii

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO2 fixation. Competitive inhibition by O2 and the loss of fixed carbon through oxygenase activity limit photosynthetic productivity. Consequently, Rubisco has been viewed as a potential target for improving catalytic efficiency and CO 2/O2 specificity as a means for improving agricultural productivity. However, it has been difficult to deduce ways to improve the enzyme because the active-site residues are nearly 100% conserved. Nonetheless, Rubisco enzymes from different species have different catalytic properties, and with a greater understanding of the structural basis for these differences, it may be possible to engineer a “better” Rubisco. In a previous study, the comparison of large-subunit sequences from ∼500 land-plants and the green alga Chlamydomonas reinhardtii identified a small set of residues that differed in regions previously shown by mutant screening to influence CO2/O2 specificity. A loop-6 amino-acid substitution, V331A, was complemented by a T342I or G344S substitution. In the present study, classical genetics, directed mutagenesis, and structural analysis identified additional residues in the loop-6 region and carboxy terminus that influence CO2/O2 specificity, indicating that subtle interactions in the loop-6 region and the carboxy terminus may be important determinants of catalytic efficiency. Ten residues in the loop-6 region differ between Chlamydomonas and land plants. Because it is difficult to analyze 10 substitutions in all possible combinations, these were combined into three groups based on their location in the X-ray crystal structure of Chlamydomonas Rubisco. Whereas loop-6 (L326I/V341I/M349L) and base-of-loop-6 (M375L/A398S/C399V) substitutions had little effect on catalytic properties, the carboxy terminal substitutions (D470P/T471A/I472M/K474T) caused a small but significant increase in CO2/O2 specificity. Despite this positive change, the other kinetic properties of the carboxy-terminal mutant enzyme remain quite different from the land-plant enzyme. Combining all 10 substitutions resulted in the loss of Rubisco holoenzyme in vivo. Thus, there must be additional residues that differ between Chlamydomonas and land plants that complement the 10 substitutions. These divergent and more-distant residues may account for differences in catalytic properties

Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO2 fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit α/β-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO2/O2 specificity, despite the fact that land-plant Rubisco has greater specificity than the Chlamydomonas enzyme. Because the residues are close to a variable loop between β-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When largesubunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO2/O2 specificity to the normal level. Further substitution with the shorter βA–βB loop of the spinach small subunit caused a 12–17% increase in specificity. The enhanced CO2/O2 specificity of the mutant enzyme is lower than that of the spinach enzyme, but the carboxylation and oxygenation kinetic constants are nearly indistinguishable from those of spinach and substantially different from those of Chlamydomonas Rubisco. Thus, this interface between large and small subunits, far from the active site, contributes significantly to the differences in catalytic properties between algal and land-plant Rubisco enzymes

Selection of Cyanobacterial (Synechococcus sp. Strain PCC 6301) RubisCO Variants with Improved Functional Properties That Confer Enhanced CO2-Dependent Growth of Rhodobacter capsulatus, a Photosynthetic Bacterium

RubisCO catalysis has a significant impact on mitigating greenhouse gas accumulation and CO2 conversion to food, fuel, and other organic compounds required to sustain life. Because RubisCO-dependent CO2 fixation is severely compromised by oxygen inhibition and other physiological constraints, improving RubisCO’s kinetic properties to enhance growth in the presence of atmospheric O2 levels has been a longstanding goal. In this study, RubisCO variants with superior structure-functional properties were selected which resulted in enhanced growth of an autotrophic host organism (R. capsulatus), indicating that RubisCO function was indeed growth limiting. It is evident from these results that genetically engineered RubisCO with kinetically enhanced properties can positively impact growth rates in primary producers.Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a ubiquitous enzyme that catalyzes the conversion of atmospheric CO2 into organic carbon in primary producers. All naturally occurring RubisCOs have low catalytic turnover rates and are inhibited by oxygen. Evolutionary adaptations of the enzyme and its host organisms to changing atmospheric oxygen concentrations provide an impetus to artificially evolve RubisCO variants under unnatural selective conditions. A RubisCO deletion strain of the nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus was previously used as a heterologous host for directed evolution and suppressor selection studies that led to the identification of a conserved hydrophobic region near the active site where amino acid substitutions selectively impacted the enzyme’s sensitivity to O2. In this study, structural alignments, mutagenesis, suppressor selection, and growth complementation with R. capsulatus under anoxic or oxygenic conditions were used to analyze the importance of semiconserved residues in this region of Synechococcus RubisCO. RubisCO mutant substitutions were identified that provided superior CO2-dependent growth capabilities relative to the wild-type enzyme. Kinetic analyses of the mutant enzymes indicated that enhanced growth performance was traceable to differential interactions of the enzymes with CO2 and O2. Effective residue substitutions also appeared to be localized to two other conserved hydrophobic regions of the holoenzyme. Structural comparisons and similarities indicated that regions identified in this study may be targeted for improvement in RubisCOs from other sources, including crop plants

Plant-like substitutions in the large-subunit carboxy terminus of Rubisco increase CO/OSpecificity-1

Uple mutant (●). Rubisco enzymes were incubated at each temperature for 10 min, and then assayed for RuBP carboxylase activity at 25°C. Activities were normalized to the specific activities measured after the 35°C incubation (wild type, 1.7 μmol/min/mg; D470P/T471A/I472M/K474T, 1.2 μmol/min/mg).<p><b>Copyright information:</b></p><p>Taken from "Plant-like substitutions in the large-subunit carboxy terminus of Rubisco increase CO/OSpecificity"</p><p>http://www.biomedcentral.com/1471-2229/8/85</p><p>BMC Plant Biology 2008;8():85-85.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527014.</p><p></p

Plant-like substitutions in the large-subunit carboxy terminus of Rubisco increase CO/OSpecificity-0

(60 μg/lane) extracted from wild type (lane 1) and the D470P/T471A/I472M/K474T quadruple mutant (lane 2). The Rubisco large subunit (LS) and small subunit (SS) are indicated.<p><b>Copyright information:</b></p><p>Taken from "Plant-like substitutions in the large-subunit carboxy terminus of Rubisco increase CO/OSpecificity"</p><p>http://www.biomedcentral.com/1471-2229/8/85</p><p>BMC Plant Biology 2008;8():85-85.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527014.</p><p></p

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) fromRhodopseudomonas palustris

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Synthetic CO2-fixation enzyme cascades immobilized on self-assembled nanostructures that enhance CO2/O2 selectivity of RubisCO

Abstract Background With increasing concerns over global warming and depletion of fossil-fuel reserves, it is attractive to develop innovative strategies to assimilate CO2, a greenhouse gas, into usable organic carbon. Cell-free systems can be designed to operate as catalytic platforms with enzymes that offer exceptional selectivity and efficiency, without the need to support ancillary reactions of metabolic pathways operating in intact cells. Such systems are yet to be exploited for applications involving CO2 utilization and subsequent conversion to valuable products, including biofuels. The Calvin–Benson–Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) play a pivotal role in global CO2 fixation. Results We hereby demonstrate the co-assembly of two RubisCO-associated multienzyme cascades with self-assembled synthetic amphiphilic peptide nanostructures. The immobilized enzyme cascades sequentially convert either ribose-5-phosphate (R-5-P) or glucose, a simpler substrate, to ribulose 1,5-bisphosphate (RuBP), the acceptor for incoming CO2 in the carboxylation reaction catalyzed by RubisCO. Protection from proteolytic degradation was observed in nanostructures associated with the small dimeric form of RubisCO and ancillary enzymes. Furthermore, nanostructures associated with a larger variant of RubisCO resulted in a significant enhancement of the enzyme’s selectivity towards CO2, without adversely affecting the catalytic activity. Conclusions The ability to assemble a cascade of enzymes for CO2 capture using self-assembling nanostructure scaffolds with functional enhancements show promise for potentially engineering entire pathways (with RubisCO or other CO2-fixing enzymes) to redirect carbon from industrial effluents into useful bioproducts