Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is probably the best-studied plant enzyme, partly because of its great abundance (constituting 30-50% total protein in leaves) but also because of its importance in the metabolism of all cells. By catalyzing carbon dioxide fixation during photosynthesis, this enzyme is responsible for virtually all of the reduced carbon found in living organisms. Rubisco couples the inorganic and organic carbon pools on earth and is the most significant route for linking these pools together by the synthesis of carbohydrate. It is, however, a poor catalyst, having both a low affinity for carbon dioxide and a small turnover number (3s-1). Autotrophic organisms must devote a major part of their synthetic capacity to produce sufficient enzyme to sustain life. The high concentrations of Rubisco and its messenger RNA have provided many opportunities to study the molecular biology of plants. This includes gene organization and gene expression in both nuclei and chloroplasts, protein targeting to chloroplasts, molecular chaperones, enzyme assembly, and many other aspects of plant cell biology.

1. Reactions Catalyzed by Rubisco

Rubisco catalyzes the first reaction in the pathways of both photosynthesis and photorespiration (1), which are respectively its carboxylase and oxygenase activities:

Photosynthesis and photorespiration are interlocking metabolic cycles, with Rubisco determining the relative rates of carbon flow through the two pathways (Fig. 1). Rubisco requires both CO2 and Mg as obligatory cofactors, resulting in carbamylation of an active-site lysine residue by CO2 and coordination of Mg2+ (1, 2).

Figure 1. The reactions catalyzed by Rubisco. The first intermediate of catalysis is the C2,C3 cis-enediol form of ribulose-bisphosphate (ribulose 1,5-P2) after abstraction of the C3 proton. The enediol can partition a number of ways, the majority into the products of carboxylation (upper reactions) or oxygenation (lower reactions). However, a number of misprotonated isomers of ribulose 1,5-P 2, for example, xylulose-bisphosphate, have been detected with the wild- type enzyme that are produced in quantity by mutations of specific amino acid residues involved in proton transfer. Phosphate elimination of the carbanion forms of intermediates are also produced by some mutants. R, —CHOH —CH20P02 v 3P D-glycerate, 3-phospho glycerate; 2P glycolate, 2-phosphoglycolate.

Five discrete partial reactions have been described for the carboxylation reaction (Eq. 1) of ribulose bisphosphate (3). The initial formation of a C2,C3-enediol intermediate of the ribulose bisphosphate substrate is followed by its carboxylation, where the enediol reacts with CO2 at the C2 position. The resulting six-carbon intermediate is hydrolytically cleaved to two molecules of 3-phosphoglycerate, resulting in an overall gain of carbon during photosynthesis.

In the oxygenase reaction (Eq. 2) of photorespiration, however, the enediol intermediate reacts with molecular oxygen to form a hydroperoxy derivative that breaks down to 2-phosphoglycolate and 3-phosphoglycerate. Photorespiration consumes ribulose bisphosphate, which results in no net gain of carbon, and also requires the consumption of energy to recycle the lost carbon. Partitioning of ribulose bisphosphate between the two reactions of photosynthesis and photorespiration can vary significantly between different photosynthetic organisms (4), which has stimulated interest in improving the carboxylation efficiency of crop plants.

Rubisco activity is modulated in plants by an inhibitor and an activator. The inhibitor 2′-carboxy arabinitol 1-phosphate (2CAIP) accumulates in some plants during darkness and binds to the active site of Rubisco (5, 6). 2CAIP is degraded by a specific phosphatase, which presumably allows Rubisco to function during photosynthesis in the light. Rubisco can be severely inhibited by a range of sugar bisphosphates, including substrate analogues. The enzyme Rubisco activase has the ability to relieve the inhibition caused by sugar bisphosphates (7), possibly by interacting with Rubisco and altering the affinity of the enzyme for bisphosphates.

2. Structure of Rubisco

Two distinct forms of Rubisco found in different species can be differentiated by their quaternary structures . Form I Rubisco is the most common structure found in photosynthetic bacteria and plants (Fig. 2a). It has a hexadecameric structure (L8S 8) with an equivalent number of both large (L) (50- 55-kDa) and small (S) (12-18-kDa) subunits. Form II Rubisco has a much simpler structure, containing L subunits alone. It is less efficient than the form I enzyme and is present in some photosynthetic bacteria that are sensitive to oxygen.

Figure 2. The organization of the subunits of L8 S8 Rubisco. In (A) the display is from above the fourfold axis of symmetry of the enzyme. The four L-subunit dimers are shown only schematically by a light trace of the positions of the Ca atoms of the backbone, whereas the structural elements of the S subunits have been displayed with Molscript. The four S subunits shown reside at the top of the molecule situation between each L-subunit dimer, with a loop extending into, but not obscuring, the prominent central channel of the L 8 core. In (B), the other four S subunits are found to occupy the same position at the bottom of the core.

Structural analysis of the L2 Rubisco dimer from Rhodospirillum rubrum reveals that amino acid residues essential for the function of each of the two active sites are located on both L subunits (8). Although the quaternary structure of L8S 8 Rubisco from plants is clearly more complex than the R rubrum homodimer, X-ray crystallographic studies reveal that the L8 core of L8S8 molecules can be built up from four R. rubrum-like dimers arranged around a fourfold axis (9). The eight S-subunits occupy large crevices between the ends of the L2-like dimers at the top and bottom of the L8 core, making extensive interactions with L subunits to stabilize the L 8 core. The S subunits do not contribute directly to the active site, but they do influence substrate affinities and turnover through contacts to elements that form the site (10).

3. Gene Organization and Expression

In most prokaryotes, the organization of the Rubisco S-subunit (rbcS) and L-subunit (rbcL) genes is relatively simple, with both genes usually closely linked and transcribed together. To the 5′ side of the initiator methionine residue of many rbc genes is a sequence similar to a Shine-Dalgarno site for ribosome binding, and sufficiently conserved to allow correct translation when cloned into Escherichia coli (11, 12).

Plant cells compartmentalize Rubisco in chloroplasts, but the genetic information is shared between chloroplast and nucleus. The rbcL genes are present in chloroplast DNA (13), and their transcription and translation in plastids uses sequences that are similar to those found in prokaryotes (14, 15), to the extent of allowing direct expression when transferred to E. coli (16). The S-subunit genes are located on nuclear chromosomes and have a more complex structural arrangement. The rbcS genes contain introns and are present as small multigene families that are often closely linked (17). Light-induced expression is mediated by both phytochrome and blue light photoreceptors (18), and positive and negative regulatory sequences are located in cis-acting transcriptional control regions. The rbcS promoters also appear to contain nuclear matrix attachment regions (MARs) (19), which may be important for their expression. The highest level of rbcS mRNA is found in leaves, but it is also found in the photosynthetic tissues in stems, petals and pods. S subunits are synthesized on free cytoplasmic polyribosomes as precursor molecules with an ^-terminal transit peptide (20). The S-subunit precursors are imported post-translationally into chloroplasts in a process requiring ATP, and the transit peptide is removed (21, 22).

4. Rubisco Assembly

Following import into chloroplasts and removal of the transit peptide, mature S subunits are assembled with chloroplast-synthesized L subunits to give the active L8S8 Rubisco holoenzyme (21, 22). This assembly process requires the assistance of another chloroplast protein (23) now known as chaperonin 60 (cpn60) (24, 25). In fact, studies on the assembly of Rubisco in chloroplasts and bacteria (23, 26, 27) led to the discovery of the molecular chaperone cpn60 and its role in the correct folding of Rubisco and many other proteins (24, 25, 28). Productive folding of Rubisco requires Mg , ATP hydrolysis, and a smaller cochaperonin molecule (29). Cpn60-mediated folding of Rubisco in bacteria uses a cochaperonin oligomer with 10-kDa subunits (24), but the folding of Rubisco in chloroplasts seems to involve a co-chaperonin oligomer with 21-kDa subunits (30). The reason for this larger cochaperonin in chloroplasts and the mechanistic details of the Rubisco assembly process in plants are currently under investigation.