RuBisCo

Figure 4.8 The active site in all a/p barrels is in a pocket formed by the loop regions that connect the carboxy ends of the p strands with the adjacent a helices, as shown schematically in (a), where only two such loops are shown, (b) A view from the top of the barrel of the active site of the enzyme RuBisCo (ribulose bisphosphate carboxylase), which is involved in CO2 fixation in plants. A substrate analog (red) binds across the barrel with the two phosphate groups, PI and P2, on opposite sides of the pocket. A number of charged side chains (blue) from different loops as welt as a Mg ion (yellow) form the substrate-binding site and provide catalytic groups. The structure of this 500 kD enzyme was determined to 2.4 A resolution in the laboratory of Carl Branden, in Uppsala, Sweden. (Adapted from an original drawing provided by Bo Furugren.)...

Figure 18.4 The hanging-drop method of protein crystallization, (a) About 10 pi of a 10 mg/ml protein solution in a buffer with added precipitant—such as ammonium sulfate, at a concentration below that at which it causes the protein to precipitate—is put on a thin glass plate that is sealed upside down on the top of a small container. In the container there is about 1 ml of concentrated precipitant solution. Equilibrium between the drop and the container is slowly reached through vapor diffusion, the precipitant concentration in the drop is increased by loss of water to the reservoir, and once the saturation point is reached the protein slowly comes out of solution. If other conditions such as pH and temperature are right, protein crystals will occur in the drop, (b) Crystals of recombinant enzyme RuBisCo from Anacystis nidulans formed by the hanging-drop method. (Courtesy of Janet Newman, Uppsala, who produced these crystals.)...

Figure 18.5 Schematic view of a diffraction experiment, (a) A narrow beam of x-rays (red) is taken out from the x-ray source through a collimating device. When the primary beam hits the crystal, most of it passes straight through, but some is diffracted by the crystal. These diffracted beams, which leave the crystal in many different directions, are recorded on a detector, either a piece of x-ray film or an area detector, (b) A diffraction pattern from a crystal of the enzyme RuBisCo using monochromatic radiation (compare with Figure 18.2b, the pattern using polychromatic radiation). The crystal was rotated one degree while this pattern was recorded.

Rubisco exists in three forms an inactive form designated E a carbamylated, but inactive, form designated EC and an active form, ECM, which is carbamylated and has Mg at its active sites as well. Carbamylation of rubisco takes place by addition of COg to its Lys ° e-NHg groups (to give e—NH—COO derivatives). The COg molecules used to carbamylate Lys residues do not become substrates. The carbamylation reaction is promoted by slightly alkaline pH (pH 8). Carbamylation of rubisco completes the formation of a binding site for the Mg that participates in the catalytic reaction. Once Mg binds to EC, rubisco achieves its active CM form. Activated rubisco displays a Ai, for CO2 of 10 to 20... 

Substrate RuBP binds much more tightly to the inactive E form of rubisco (An = 20 nM) than to the active ECM form (A, for RuBP = 20 ixM). Thus, RuBP is also a potent inhibitor of rubisco activity. Release of RuBP from the active site of rubisco is mediated by rubisco activase. Rubisco activase is a regulatory protein it binds to A-form rubisco and, in an ATP-dependent reaction, promotes the release of RuBP. Rubisco then becomes activated by carbamylation and Mg binding. Rubisco activase itself is activated in an indirect manner by light. Thus, light is the ultimate activator of rubisco. 

As discussed in Section 22.7, illumination of chloroplasts leads to light-driven pumping of protons into the thylakoid lumen, which causes pH changes in both the stroma and the thylakoid lumen (Figure 22.27). The stromal pH rises, typically to pH 8. Because rubisco and rubisco activase are more active at pH 8, COg fixation is activated as stromal pH rises. Fructose-1,6-bisphosphatase, ribulose-5-phosphate kinase, and glyceraldehyde-3-phosphate dehydrogenase all have alkaline pH optima. Thus, their activities increase as a result of the light-induced pH increase in the stroma. 

Eormadon of PEP by pyruvate Pi dikinase reini-dates the cycle. The CO9 liberated in the bundle slieadi cell is used to syndiesize hexose by die convendonal rubisco-Calvin cycle series of reacdons. 

The amino acid composition of storage proteins differs from that of the complete sprout [12, 13]. At least in the case of oilseed rape, alfalfa (Medicago sativa L.) and Camelina sativa, amino acids in the sprout are used mainly, either directly or indirectly, for the synthesis of the Rubisco proteins. Computer analysis shows that the amino acid composition of cruciferin and napin is completely different to the amino acid composition of Rubisco. This indicates that amino acids released from the seed storage proteins must be converted into other amino acids prior to Rubisco synthesis. 

Fig. 3.2 Rapeseeds were germinated from 12to 168 h in airlift tank. The total soluble proteins were extracted and separated by 15% SDS-PAGE. The gel was stained with Coomassie blue. Between 36 and 60 h, the degradation of storage proteins and the de novo synthesis of Rubisco is clearly visible.

Fig. 3.3 A. Northern blot showing the synthesis of Rubisco SSU mRNA after 24-36 h of sprouting in an airlift tank. Total RNA was isolated from sprouts germinated from 12 to 168 hours. B. On the same filter, unlabelled Rubisco RNA produced by in vitro transcription was loaded as a control. The amount of control RNA is indicated in pg.

Transcriptional inhibitors could be used simultaneously. Rifampicin blocks chloroplast and mitocondrian RNA synthesis [23, 24], while tagetitoxin is a very specific inhibitor of chloroplast RNA polymerase [25]. Treatment with these antibiotics does not inhibit Rubisco SSU synthesis since the promoter is part of the nuclear genome, while the cytosolic ribosomes are not affected by streptomycin. Therefore SSU promoters can be used to drive transgene expression and facilitate the accumulation of recombinant proteins. Expressed proteins are targeted to a suitable cellular compartment, such as the cytoplasm, apoplastic space or chloroplast, depending on the nature of the protein. 

As discussed above, Rubisco levels have been reduced by expressing antisense RNA in transgenic tobacco plants [26]. Plants expressing antisense rbcS RNA showed reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced levels of LSU and SSU proteins. 

We have isolated Rubisco small subunit promoters from several plant species and tested their strength with gusA and ALB (human serum albumin) transgenes in sprouts. The highest level of expression in Brassica napus sprouts has been obtained... 

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