Advancing Our Understanding and Capacity to Engineer Nature’s CO2-Sequestering Enzyme, Rubisco

There is a growing impetus in developing novel strategies to address global concerns regarding food security. As crop productivity gains through traditional breeding begin to lag and arable land becomes scarcer, it seems that we are heading for unsustainable global populations. It has been foreshadowed that global food production will need to rise more than 50% before 2050 to meet the ever-increasing demand. Compounding the problem are the uncertainties of climate change and its impact on agriculture. Strategies to improve crop yield potential have begun to examine aspects of supercharging photosynthesis to drive a new “green revolution.” Central to many of these strategies is addressing the limitation of nature’s CO2-fixing enzyme, Rubisco. The catalytic incorporation of CO2 into ribulose 1,5-bisphosphate (RuBP) by Rubisco is the first step in the production of carbohydrates by plants, which are used to build biomass and produce energy during growth and development. Despite the pivotal role of Rubisco in linking the inorganic (CO2) and the organic (biomass) phases of the global carbon cycle, it is a slow and confused catalyst, limiting productivity and resource (e.g. water and nutrients) use efficiency in many plants (Long et al., 2006). Understandably, Rubisco has been studied intensively and is a prime target for genetic engineering to improve photosynthetic efficiency (Raines, 2006; Parry et al., 2007). Although the challenge of making a “better Rubisco” has exceeded the grasp and career of many scientists, recent advances indicate that it is not insurmountable. Here, we examine conceptual and technological breakthroughs over the last decade that have identified new and unconventional members of the Rubisco family, revealed molecular aspects of Rubisco biogenesis in plastids and cyanobacteria, advanced our understanding of its catalytic chemistry, and widened our appreciation of the challenges we face to improve Rubisco activity and plant productivity. Rubisco is an ancient enzyme, its history beginning more than 3.5 billion years ago, when Earth’s atmosphere was high in CO2 and before the origins of oxygen (O2)-producing photosynthesis (Fig. 1). Modern genomic sequencing projects have identified Rubisco and Rubisco-like proteins (RLPs) in organisms from three kingdoms of life, with some microorganisms possessing multiple forms of these enzymes (Andersson and Backlund, 2008). Phylogenetic analysis of these sequences supports the existence of three different clades of Rubisco (denoted forms I, II, and III), which, together with the more diverse RLPs (or form IV Rubisco), probably share a common ancestor, most likely that of a methanogenic archaea (Fig. 1; Tabita et al., 2008). Structurally, the RLPs lack key conserved active-site residues of Rubiscos and, therefore, do not inherently bind RuBP or catalyze CO2 fixation. Detailed studies of some RLPs indicate that they participate in thiosulfate oxidation, catalyzing the enolization of a RuBP analog (Saito et al., 2009). The increasing variety of sequenced genomes has led to the identification of a growing number of RLPs in multiple bacterial lineages, suggesting that these proteins may have other catalytic functions. Clearly, our perception of RLP biology is still in its infancy. Hypothetical profiles of Rubisco phylogeny, the evolutionary timelines of different photosynthetic organisms, and variation in atmospheric CO2 (thicker line) and O2 levels during earth’s history. Hypothetical atmospheric CO2 and O2 levels prior to 0.6 billion years ago are represented by dotted lines. Quaternary structures of each Rubisco were drawn with Pymol using Protein Data Bank coordinates for the spinach (Spinacia oleracea) (L2)4S8 (8RUC), R. rubrum L2 (5RUB), Pyrococcus horikoshii (L2)4 (2CWX), and Thermococcus kodakaraensis (L2)5 (1GEH) enzymes. Structures for larger form II (L2)n Rubiscos are unavailable. Circular images depict types of organisms where the different Rubisco forms are found. Figure details were adapted from Tabita et al. (2008) and Badger et al. (2002). From a structural point of view, all Rubisco enzymes comprise at least two large (L-) subunits of approximately 50 kD. Despite there being as little as 30% amino acid identity between the different Rubisco forms, they all show a conserved L-subunit structure comprising an N-terminal domain (approximately 150 amino acids) and a larger C-terminal domain (approximately 320 amino acids) that forms an α/β-barrel (Fig. 2). Paired L-subunits arrange head to tail to form a dimer (L2), with two active sites located at the L-L interface. The highly conserved catalytic residues predominantly reside within the α/β-barrel domain, with a few residues supplied by the N-terminal domain of the adjacent L-subunit. Conserved structural features of Rubisco. A, Spinach L8S8 Rubisco (Protein Data Bank 8RUC) drawn using Pymol to highlight arrangement of the S-subunits (blue) capping the catalytic core of four L2 subunits (green). B, Structural details for one L-subunit of an L2 pair highlighting one active site within the α/β-barrel of the C-terminal domain (green ribbons) and residues in the N-terminal domain (yellow ribbons) that contribute to the second active site in each L2. C, Arrangement of the conserved Rubisco active-site residues within a L-subunit C-terminal domain relative to carbamylated Lys-201 (K201X), bound Mg2+, and the six-carbon reaction intermediate mimic, 2-carboxyarabinitol 1,5-bisphosphate (CABP). The activating CO2 in K201X and the approximate positioning of substrate CO2 that binds to C-2 of the RuBP enediol are highlighted in ball-and-stick representations. Residues are numbered relative to spinach Rubisco. The conserved active site residues Glu-60 and Asn-123 from the N-terminal domain of the paired L-subunit are not shown. During evolution, different organisms have developed diverse arrangements of the Rubisco L2 building blocks. Form I Rubiscos are the most abundant form found in plants, algae, and many photosynthetic bacteria (Fig. 1). The L2 subunits in form I Rubiscos are arranged in an (L2)4 core, with two groups of four small (S-) subunits (approximately 13–17 kD) capping the L8 core to form an L8S8 molecule (Fig. 2A). Although not strictly required for CO2 fixation, the S-subunits are essential for maximal activity and provide structural stability (Andersson and Backlund, 2008). Recent evidence suggests that S-subunits in the unicellular green alga Chlamydomonas reinhardtii may also play a structural role in pyrenoid development (Genkov et al., 2010). Rubiscos classed as forms II and III lack S-subunits, containing only L-subunits arranged into L2 to (L2)5 complexes (Fig. 1). These Rubiscos are primarily found in phototrophic proteobacteria, chemoautotrophs, dinoflagellates, and achaea. Although the classification of Rubisco enzymes into forms I, II, and III is generally supported by sequence phylogenies, quaternary structures, and functional properties, there are exceptions. For example, the structural and biochemical properties of the Rubisco enzyme from the archaea Methanococcoides burtonii correlate to form III, despite closer sequence identity to form II Rubiscos (Alonso et al., 2009). Like the RLPs, further discovery and characterization of divergent Rubisco forms are of great importance if we are to understand the diversity of the Rubisco family, refine its nomenclature, and comprehend its origin and evolution. Despite amino acid sequence variability within the Rubisco family, key active-site residues are absolutely conserved among forms I, II, and III Rubiscos (Andersson and Backlund, 2008). As a result, the activation process and complex catalytic chemistry are also preserved, despite the different biological roles of forms I and II Rubiscos, which initiate primary carbon assimilation, and the catabolic role of archaeal form III enzymes, which remove RuBP produced during purine/pyrimidine metabolism (Sato et al., 2007). Most structure-function studies have focused on Rubiscos involved in carbon assimilation and provide much of our mechanistic understanding of its catalysis (for review, see Parry et al., 2003). Prior to catalysis, Rubisco needs to be preactivated via the reaction of a CO2 molecule with a conserved active-site Lys (residue 201 in most plant Rubisco L-subunits) to form a carbamate, which is then stabilized by Mg2+ binding (Fig. 2C; Andersson and Backlund, 2008). Following activation, Rubisco can productively bind RuBP and catalyze a complex five-step reaction that adds a CO2 and a water molecule to RuBP, followed by its cleavage and release of two 3-phosphoglycerate (3PGA) molecules (Fig. 3). The complexity of the multistep process can lead to unwanted side reactions that result in the formation of inhibitors such as xyulose-1,5-bisphosphate (Pearce, 2006; Parry et al., 2007). Furthermore, the electrostatic similarity between O2 and CO2 and their disproportionate atmospheric abundance (21% O2, 0.04% CO2) make it hard for Rubisco to totally discriminate between them, resulting in the unwanted oxygenation of RuBP and the production of one molecule of 3PGA and one of 2-phosphoglycolate. In plants, 2-phosphoglycolate is recycled back to 3PGA via photorespiration, an energy-consuming process (e.g. ATP) that liberates fixed carbon as CO2 (Peterhansel et al., 2008; Fig. 3). Simplified scheme illustrating how CO2 fixed to RuBP by Rubisco is distributed among the resulting two molecules of 3PGA that feed into the photosynthetic Calvin cycle to produce triose phosphates (glyceraldehyde 3-phosphate [G3P]) for carbohydrate synthesis or RuBP regeneration. The contrasting oxygenation reaction of Rubiscos produces 2-phosphoglycolate (2PG), which requires the photorespiratory pathway to recycle it back to 3PGA. Photorespiration is a complex pathway that involves four subcellular compartments and multiple enzymatic steps (represented by dashed lines), requires additional energy (ATP), and results in a loss of fixed CO2 in the mitochondria (Maurino and Peterhansel, 2010). Detailed understanding of Rubisco catalysis has taken advantage of the exponential rise in computer power and the increasing accuracy of theoretical protein models. Several groups have applied up-to-date computational tools to examine the energetics and atomistic details of Rubisco’s carboxylation and oxygenation reactions (Kannappan and Gready, 2008). While these calculations reveal molecular details of the contribution of active-site residues to Rubisco catalysis, the large size of Rubisco prevents the inclusion of all atoms in the calculation. As a result, the design of “better” Rubiscos using in silico modeling tools remains to be demonstrated. The Rubisco family shows significant catalytic variability despite sharing the same catalytic chemistry (Fig. 4). Unfortunately, comprehensive catalytic studies have only been made for relatively few Rubiscos (Supplemental Table S1), limiting our capacity to fully appreciate the connections between catalytic and sequence diversity and the influence of temperature on the activity of evolutionarily diverse Rubiscos. As highlighted by Tcherkez et al. (2006), improvements in CO2 fixation rate for forms I and II Rubiscos generally come at the expense of affinity for CO2. Therefore, photosynthetic organisms that live under high CO2 and low O2 (e.g. proteobacteria such as Rhodospirillum rubrum) or that have evolved complex, energy-expensive, biochemical CO2-concentrating mechanisms that elevate CO2 levels around Rubisco, as seen in C4 plants, many algae, and cyanobacteria, have Rubiscos with lower CO2 affinities (i.e. higher K m for CO2) but higher carboxylation rates (v CO2; Fig. 4, A and B). This is usually accompanied by lower specificities for CO2 over O2 (SC/O; Fig. 4C). In contrast, C3 plants (which include most crops) and algae that lack a CO2-concentrating mechanism have higher CO2 affinities, better CO2/O2 specificities, but slower carboxylation rates. Comparative catalytic features of different Rubisco forms measured at 25°C. Individual dashes in each column represent separate catalytic measurements for each Rubisco form (detailed in Supplemental Table S1). Yellow circles indicate the catalytic measurements for green algal Rubisco. SC/O values are calculated as (v CO2/K m O2)/(v O2/K m CO2), where v CO2 and v O2 are the maximum rates of RuBP carboxylation and oxygenation and K m O2 and K m CO2 are the apparent K m values for O2 and CO2, respectively. A growing realization is that the catalytic diversity of Rubiscos originates from residues distant from the active site and generally not conspicuous in the more than 20 different Rubisco x-ray structures currently available (Andersson and Backlund, 2008). The significant catalytic diversity observed in Rubiscos from diverse C4 and C3 species (Galmes et al., 2005; Ghannoum et al., 2005; Kubien et al., 2008; Carmo-Silva et al., 2010) has prompted the use of bioinformatic analyses to identify potential “catalytic switches,” key residues responsible for the faster v CO2 rates of C4 Rubiscos (Christin et al., 2008). This approach has yet to be applied to unveil residues responsible for the variation in CO2/O2 specificity of Rubiscos of related C3 species (Galmes et al., 2005). Confidence that catalytic improvements of C3 Rubiscos are possible stems from the finding that some red algae have the most efficient Rubiscos (Tcherkez et al., 2006; Parry et al., 2007). Indeed, the successful transfer of the catalytic properties of the Rubisco enzyme from the red alga Griffithsia monilis into a C3 crop has the potential to raise yields by approximately 30% (Long et al., 2006) as a result of higher discrimination between CO2 and O2 and reduced photorespiration (Fig. 3). The carbon losses due to photorespiration are even larger at elevated temperatures, which favor increasing the relative oxygenation activity of Rubisco (Sage, 2002). Key to developing strategies to engineer Rubisco is a better understanding of its biogenesis and regulation. The mechanism and requirements that coordinate the expression and assembly of L- and S-subunits into L8S8 Rubisco in chloroplasts are still rudimentary (Fig. 5; Nishimura et al., 2008). In prokaryotes and the plastid genome of nongreen algae, genes for the L- (rbcL) and S- (rbcS) subunits of form I Rubiscos are colocated in an operon. In higher plants and green algae, the single rbcL gene remains by the plastid multiple of the gene are located in the (Fig. The by which expression of the and L- and S-subunits, are remains Recent evidence suggests that L-subunit expression may be by the of synthesis L-subunit bind to the rbcL to its and 2007). The importance of Rubisco synthesis in plastids is as it is produced in high to for its slow and enzymatic For example, in C3 plants, between and 30% of the protein (i.e. approximately of the is in Rubisco. The complexity of Rubisco biogenesis and its by in plant and and molecular by genes in the as are highlighted in for details of the and the challenges in the L8S8 Figure details were adapted from Nishimura et al. in the biogenesis process are by gene and and complexes and and understanding of Rubisco biogenesis is As with other synthesis of the in the an N-terminal for transfer the of molecular and then the complexes (Fig. 5; 2008). the the S-subunits further prior to assembly into L8S8 complexes et al., of L-subunits in the with the of rbcL via a that requires a variety of et al., A conserved has been identified in Chlamydomonas and that binds to the of rbcL to the of the et al., 2010). The aspects of rbcL and in chloroplasts also lack but to show to the bacterial with its on for (for review, see Nishimura et al., 2008). L-subunits are for N-terminal (for review, see et al., that with the of by and then and via an an N-terminal that is by an In some is also by a Rubisco L-subunit Recent analysis revealed a large between Rubisco L-subunit and the and N-terminal of L-subunit in L8S8 Rubisco but not the S-subunits et al., suggesting that of L2 assembly and prior to assembly (Fig. Although the of these it is that they plant Rubisco from et al., 2008). In it is that L-subunits with the and the (for review, see Nishimura et al., 2008). These and the to the of the complexes of the and with L8S8 Rubisco have that L-subunits with a gene is located between rbcL and in et al., et al., 2010). the assembly of L-subunits into (L2)4 complexes and are then by the binding of S-subunits that produce the L8S8 In where is located separate from the of has on Rubisco synthesis et al., 2006). This the of the in higher plants has a functional role in Rubisco assembly in plastids (Fig. to Rubisco in higher plants and green algae may also need to the for Rubisco’s protein Rubisco Fig. This protein the energy of to remove active-site bound which are produced to Rubisco activity as or as the result of reactions (e.g. 2006; Parry et al., 2008). The to a structure of to our understanding of how of subunits with Rubisco. A that amino between and of the located on the of form I Rubiscos, with residues in the C-terminal domain of et al., 2008). of the domain is to L-subunit that result in the of that over the active site RuBP release (Fig. breakthroughs in the understanding of Rubisco biology and its influence on with technological advances in its genetic have pivotal into structure-function and its catalytic These breakthroughs have the challenge of the catalytic properties of Rubisco and their on photosynthesis and plant between catalytic catalytic chemistry and the many molecular requirements may have the capacity of many Rubiscos to catalytic and 2008). The of photosynthetic organisms on Rubisco its high expression and its to with many molecular (Fig. may have its the of Rubisco’s complex catalytic chemistry under high CO2 and O2 (Fig. and the CO2 binds to the RuBP enediol a complex with Rubisco, making it to it from O2 (Kannappan and Gready, may large catalytic the diversity of Rubiscos that there is for (Fig. and the of how Rubiscos in nongreen algae have evolved further and sequence are more in nongreen algae the genes for Rubisco subunits and their molecular and are all located within the Rubiscos may not be with a for by (Pearce, 2006). During the last the of protein has a for and and 2008). the of a of followed by a process to identify proteins with the For Rubisco, a variety of using photosynthetic and have been developed (for review, see and with most that improve Rubisco and assembly there are where of reinhardtii and Rubisco that show improvements in catalytic (i.e. v CO2, K m CO2, and have been et al., et al., 2010). In one catalytic improvements have been found to be to Rubisco in et al., how these improvements to in photosynthesis and plant growth The of plant Rubisco L-subunits to form in to such studies to in genetic or genome are applied to organisms where genetic engineering tools are most such as the unicellular alga Chlamydomonas and the C3 plant Chlamydomonas is a engineering as can be made to rbcL and It has been used to show the importance of the in Rubisco catalytic efficiency via of L- and S- subunits and for the of Rubiscos comprising higher plant S-subunits with Chlamydomonas L-subunits et al., 2005; et al., 2010). Phylogenetic and structural of Rubiscos have identified L- and residues that Several of these sites and have been using roles for and in the L-subunit and the between A and of the and et al., 2007). to and better Rubiscos will be on the Chlamydomonas engineering the L8S8 Rubisco in is by the of rbcL and in different genomes (Fig. Recent of Rubisco in have focused on rbcL by and have the of higher plant Rubisco with bacterial R. rubrum and burtonii Rubiscos and et al., 2009). This has also the of L8S8 Rubiscos comprising L-subunits and S-subunits, which show catalytic et al., 2008). In each the growth and photosynthetic properties of the plants have with the and catalytic properties of the Rubisco, the accuracy of the used to photosynthetic carbon and studies in have also highlighted to Rubisco efficient Rubiscos from red algae be produced in plant plastids due to evolutionary in their and assembly requirements et al., with assembly of L-subunits with S-subunits also their assembly into L8S8 complexes et al., 2008). and in the L-subunit were also found to L8S8 synthesis et al., et al., the that with molecular during Rubisco biogenesis the sequence with engineering the in have also with the apparent for S-subunits to of S-subunits and has been by the of the using plant et al., The of assembly by the S- and L-subunits together with has also been et al., the of for engineering or Rubiscos in plastids has yet to be demonstrated. Recent that CO2 can crop yield for the that in photosynthesis can improve yield potential (Long et al., 2006; et al., 2007). photosynthetic rates in C4 plants lead to more biomass being produced for a of relative to C3 are key strategies to photosynthesis in C3 plants and improve crop yield The point of these strategies is to the catalytic of Rubisco by the process found in C4 plants, which CO2 around Rubisco to photorespiration and its energy and carbon loss (Fig. 3). the last C4 plants have evolved a variety of CO2-concentrating strategies that their Rubiscos to with lower CO2 affinities CO2 fixation rates (Fig. 4). As a result, C4 plants high photosynthetic rates with Rubisco use and can under low CO2 the need for water loss et al., 2005). A variety of strategies for CO2-concentrating into C3 plants to photorespiration are under (Peterhansel et al., 2008; and Peterhansel, 2010). These to features into improve productivity by proteins from into or engineer new into plastids that photorespiration and release CO2 in the While each challenges in their and into further in yields and in water and use will likely the lead of C4 plants by increasing the v CO2 of the C3 Rubisco. The are available in the of Supplemental Table properties for different Rubisco forms at 25°C.