Trends in Research and Development for CO2 Capture and Sequestration.

Technological and medical advances over the past few decades epitomize human capabilities. However, the increased life expectancies and concomitant land-use changes have significantly contributed to the release of ∼830 gigatons of CO2 into the atmosphere over the last three decades, an amount comparable to the prior two and a half centuries of CO2 emissions. The United Nations has adopted a pledge to achieve "net zero", i.e., yearly removing as much CO2 from the atmosphere as the amount emitted due to human activities, by the year 2050. Attaining this goal will require a concerted effort by scientists, policy makers, and industries all around the globe. The development of novel materials on industrial scales to selectively remove CO2 from mixtures of gases makes it possible to mitigate CO2 emissions using a multipronged approach. Broadly, the CO2 present in the atmosphere can be captured using materials and processes for biological, chemical, and geological technologies that can sequester CO2 while also reducing our dependence on fossil-fuel reserves. In this review, we used the curated literature available in the CAS Content Collection to present a systematic analysis of the various approaches taken by scientists and industrialists to restore carbon balance in the environment. Our analysis highlights the latest trends alongside the associated challenges.

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO2 Fixation.

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO2 via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure-function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

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.

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.IMPORTANCE 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.

Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides, metabolically versatile members of 'Proteobacteria' from aquatic and soil habitats.

RubisCO, the CO2 fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes 12 CO2 faster than 13 CO2 resulting in 13 C-depleted biomass, enabling the use of δ13 C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ13 C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the 'Proteobacteria' Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ13 C values in nature.

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

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.

RubisCO selection using the vigorously aerobic and metabolically versatile bacterium Ralstonia eutropha.

UNLABELLED: Recapturing atmospheric CO2 is key to reducing global warming and increasing biological carbon availability. Ralstonia eutropha is a biotechnologically useful aerobic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) for CO2 utilization, suggesting that it may be a useful host to bioselect RubisCO molecules with improved CO2 -capture capabilities. A host strain of R. eutropha was constructed for this purpose after deleting endogenous genes encoding two related RubisCOs. This strain could be complemented for CO2 -dependent growth by introducing native or heterologous RubisCO genes. Mutagenesis and suppressor selection identified amino acid substitutions in a hydrophobic region that specifically influences RubisCO's interaction with its substrates, particularly O2 , which competes with CO2 at the active site. Unlike most RubisCOs, the R. eutropha enzyme has evolved to retain optimal CO2 -fixation rates in a fast-growing host, despite the presence of high levels of competing O2 . Yet its structure-function properties resemble those of several commonly found RubisCOs, including the higher plant enzymes, allowing strategies to engineer analogous enzymes. Because R. eutropha can be cultured rapidly under harsh environmental conditions (e.g., with toxic industrial flue gas), in the presence of near saturation levels of oxygen, artificial selection and directed evolution studies in this organism could potentially impact efforts toward improving RubisCO-dependent biological CO2 utilization in aerobic environments. ENZYMES: d-ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39; phosphoribulokinase, EC 2.7.1.19.

RubisCO of a nucleoside pathway known from Archaea is found in diverse uncultivated phyla in bacteria.

Metagenomic studies recently uncovered form II/III RubisCO genes, originally thought to only occur in archaea, from uncultivated bacteria of the candidate phyla radiation (CPR). There are no isolated CPR bacteria and these organisms are predicted to have limited metabolic capacities. Here we expand the known diversity of RubisCO from CPR lineages. We report a form of RubisCO, distantly similar to the archaeal form III RubisCO, in some CPR bacteria from the Parcubacteria (OD1), WS6 and Microgenomates (OP11) phyla. In addition, we significantly expand the Peregrinibacteria (PER) II/III RubisCO diversity and report the first II/III RubisCO sequences from the Microgenomates and WS6 phyla. To provide a metabolic context for these RubisCOs, we reconstructed near-complete (>93%) PER genomes and the first closed genome for a WS6 bacterium, for which we propose the phylum name Dojkabacteria. Genomic and bioinformatic analyses suggest that the CPR RubisCOs function in a nucleoside pathway similar to that proposed in Archaea. Detection of form II/III RubisCO and nucleoside metabolism gene transcripts from a PER supports the operation of this pathway in situ. We demonstrate that the PER form II/III RubisCO is catalytically active, fixing CO2 to physiologically complement phototrophic growth in a bacterial photoautotrophic RubisCO deletion strain. We propose that the identification of these RubisCOs across a radiation of obligately fermentative, small-celled organisms hints at a widespread, simple metabolic platform in which ribose may be a prominent currency.

Functional metagenomic selection of ribulose 1, 5-bisphosphate carboxylase/oxygenase from uncultivated bacteria.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO2 /O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2 -fixing enzymes not previously characterized.

Structure-function studies with the unique hexameric form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodopseudomonas palustris.

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a "closed" conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile(165) and Met(331)) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala(47)) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.

A Rubisco mutant that confers growth under a normally "inhibitory" oxygen concentration.

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) is a globally significant biocatalyst that facilitates the removal and sequestration of CO2 from the biosphere. Rubisco-catalyzed CO2 reduction thus provides virtually all of the organic carbon utilized by living organisms. Despite catalyzing the rate-limiting step of photosynthetic and chemoautotrophic CO2 assimilation, Rubisco is markedly inefficient as the competition between O2 and CO2 for the same substrate limits the ability of aerobic organisms to obtain maximum amounts of organic carbon for CO2-dependent growth. Random and site-directed mutagenesis procedures were coupled with genetic selection to identify an "oxygen-insensitive" mutant cyanobacterial (Synechococcus sp. strain PCC 6301) Rubisco that allowed for CO2-dependent growth of a host bacterium at an oxygen concentration that inhibited growth of the host containing wild-type Synechococcus Rubisco. The mutant substitution, A375V, was identified as an intragenic suppressor of D103V, a negative mutant enzyme incapable of supporting autotrophic growth. Ala-375 (Ala-378 of spinach Rubisco) is a conserved residue in all form I (plant-like) Rubiscos. Structure-function analyses indicate that the A375V substitution decreased the enzyme's oxygen sensitivity (and not CO2/O2 specificity), possibly by rearranging a network of interactions in a fairly conserved hydrophobic pocket near the active site. These studies point to the potential of engineering plants and other significant aerobic organisms to fix CO2 unfettered by the presence of O2.

Plant-like substitutions in the large-subunit carboxy terminus of Chlamydomonas Rubisco increase CO2/O2 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 approxiamtely 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 and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses the key reaction by which inorganic carbon may be assimilated into organic carbon. Phylogenetic analyses indicate that there are three classes of bona fide RubisCO proteins, forms I, II and III, which all catalyse the same reactions. In addition, there exists another form of RubisCO, form IV, which does not catalyse RuBP carboxylation or oxygenation. Form IV is actually a homologue of RubisCO and is called the RubisCO-like protein (RLP). Both RubisCO and RLP appear to have evolved from an ancestor protein in a methanogenic archaeon, and comprehensive analyses indicate that the different forms (I, II, III and IV) contain various subgroups, with individual sequences derived from representatives of all three kingdoms of life. The diversity of RubisCO molecules, many of which function in distinct milieus, has provided convenient model systems to study the ways in which the active site of this protein has evolved to accommodate necessary molecular adaptations. Such studies have proven useful to help provide a framework for understanding the molecular basis for many important aspects of RubisCO catalysis, including the elucidation of factors or functional groups that impinge on RubisCO carbon dioxide/oxygen substrate discrimination.

Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.

There are four forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) found in nature. Forms I, II, and III catalyse the carboxylation and oxygenation of ribulose 1,5-bisphosphate, while form IV, also called the Rubisco-like protein (RLP), does not catalyse either of these reactions. There appear to be six different clades of RLP. Although related to bona fide Rubisco proteins at the primary sequence and tertiary structure levels, RLP from two of these clades is known to perform other functions in the cell. Forms I, II, and III Rubisco, along with form IV (RLP), are thought to have evolved from a primordial archaeal Rubisco. Structure/function studies with both archaeal form III (methanogen) and form I (cyanobacterial) Rubisco have identified residues that appear to be specifically involved with interactions with molecular oxygen. A specific region of all form I, II, and III Rubisco was identified as being important for these interactions.

Structural and functional consequences of the replacement of proximal residues Cys(172) and Cys(192) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii.

Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V(c) (V(max) for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the K(m) for CO(2) and O(2) was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 degrees C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 degrees C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of beta-strand 1 of the alpha/beta-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs.

About 30 years have now passed since it was discovered that microbes synthesize RubisCO molecules that differ from the typical plant paradigm. RubisCOs of forms I, II, and III catalyze CO(2) fixation reactions, albeit for potentially different physiological purposes, while the RubisCO-like protein (RLP) (form IV RubisCO) has evolved, thus far at least, to catalyze reactions that are important for sulfur metabolism. RubisCO is the major global CO(2) fixation catalyst, and RLP is a somewhat related protein, exemplified by the fact that some of the latter proteins, along with RubisCO, catalyze similar enolization reactions as a part of their respective catalytic mechanisms. RLP in some organisms catalyzes a key reaction of a methionine salvage pathway, while in green sulfur bacteria, RLP plays a role in oxidative thiosulfate metabolism. In many organisms, the function of RLP is unknown. Indeed, there now appear to be at least six different clades of RLP molecules found in nature. Consideration of the many RubisCO (forms I, II, and III) and RLP (form IV) sequences in the database has subsequently led to a coherent picture of how these proteins may have evolved, with a form III RubisCO arising from the Methanomicrobia as the most likely ultimate source of all RubisCO and RLP lineages. In addition, structure-function analyses of RLP and RubisCO have provided information as to how the active sites of these proteins have evolved for their specific functions.

Structural analysis of altered large-subunit loop-6/carboxy-terminus interactions that influence catalytic efficiency and CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase.

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel of ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in discriminating between CO2 and O2. Genetic screening in Chlamydomonas reinhardtii previously identified a loop-6 V331A substitution that decreases carboxylation and CO2/O2 specificity. Revertant selection identified T342I and G344S substitutions that restore photosynthetic growth by increasing carboxylation and specificity of the V331A enzyme. In numerous X-ray crystal structures, loop 6 is closed or open depending on the activation state of the enzyme and the presence or absence of ligands. The carboxy terminus folds over loop 6 in the closed state. To study the molecular basis for catalysis, directed mutagenesis and chloroplast transformation were used to create T342I and G344S substitutions alone. X-ray crystal structures were then solved for the V331A, V331A/T342I, T342I, and V331A/G344S enzymes, as well as for a D473E enzyme created to assess the role of the carboxy terminus in loop-6 closure. V331A disturbs a hydrophobic pocket, abolishing several van der Waals interactions. These changes are complemented by T342I and G344S, both of which alone cause decreases in CO2/O2 specificity. In the V331A/T342I revertant enzyme, Arg339 main-chain atoms are displaced. In V331A/G344S, alpha-helix 6 is shifted. D473E causes disorder of the carboxy terminus, but loop 6 remains closed. Interactions between a transition-state analogue and several residues are altered in the mutant enzymes. However, active-site Lys334 at the apex of loop 6 has a normal conformation. A variety of subtle interactions must be responsible for catalytic efficiency and CO2/O2 specificity.

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 CO(2) 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 alpha/beta-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 CO(2)/O(2) 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 beta-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When large-subunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO(2)/O(2) specificity to the normal level. Further substitution with the shorter betaA-betaB loop of the spinach small subunit caused a 12-17% increase in specificity. The enhanced CO(2)/O(2) 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.

Substitutions at the Asp-473 latch residue of chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO(2)/O(2) specificity.

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-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 approximately 16% 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.