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

Abstract

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