FASE II: Ejecución

The process whereby light energy is converted into chemical energy which is subsequently used to synthesize organic compounds is known as photosynthesis. Photosynthesis is the source of carbon and energy for most forms of life on earth and is undertaken by a number of groups of organisms including all green plants, the eukaryotic algae, diatoms, cyanobacteria and the purple bacteria. Photosynthesis can be divided into two phases:-

1. A light-dependent phase in which energy is used to generate a proton gradient which is used to synthesize ATP, and to produce reducing power in the form af NADPH at the expense of water oxidation.

2. A light-independent phase in which the reducing power and the ATP generated in the first phase are used for the fixation of carbon dioxide and its conversion to organic compounds.

In eukaryotic organisms the photosynthetic reactions take place in the chloroplast, and the fixation of carbon dioxide (CO2) occurs in the stromal compartment of the organelle in a series of enzyme-catalyzed reactions known as the Calvin cycle. The first step in this cycle is catalyzed by the enzyme ribulose-1.5-bisphosphate carboxylase-oxygenase (Rubisco EC 4.1.1.39; Fig. 1). This bifunctional enzyme catalyzes either the carboxylation of the compound ribulose-1.5-bisphosphate (RuBP) using CO2 and H2O to produce two molecules of 3-phosphoglycerate (PGA), or alternatively through the oxygenase reaction using one molecule of oxygen (02) rather than CO2 , to produce one molecule o f 3- phosphoglycerate and one molecule of 2-phosphoglycolate. The molecule 2-posphoglycolate is the substrate for a series of reactions that lead to the loss of carbon as carbon dioxide and this process is referred to as photorespiration (Fig. 1; for review see Ogren, 1984). The efficiency with which carbon is fixed relative to oxygen determines the growth of the organism and is determined by the properties of the Rubisco enzyme and by the relative concentrations of CO2 and O2 around the enzyme.

Figure 1. Photosynthetic an d ph otorespiratory pathw ays

The two pathways are presented schematically. Note that because the two complementary routes of glycine metabolism in the mitochondria, two molecules of 2-phosphoglycolate must enter the respiratory pathway for each molecule of serine, CO2 and NH3 by the glycine decarboxylase reaction. Symbols: RuBP, ribulose-1.5-bisphosphate; PGA, 3- phosphoglycerate; P-glycolate, 2-phosphoglycolate; OG, a-keto-oxoglutarate; OH-pyruvate, hydroxypyruvate; THF, tetrahydrofolic acid. Amino acids are shown in the three-letter code as described by IUPAC-IUB (1970; 1984). This Figure was taken from a paper by

Somerville et al. (1983).

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Laing et al. (1974) showed that oxygen is linearly competitive for the carboxylase reaction and that carbon dioxide is linearly competitive for the oxygenase reaction. Since the

early observation by Saussure in 1804 (cited in Kimball, 1983) that Pisum sativum plants

exposed to high CO2 concentrations grew better than control plants in ambient air, the effect of increased CO2 concentration on agricultural yield has been studied intensively. Recent studies have indicated that with a doubling of the atmospheric CO2 concentration the economic yield of many plants will probably increase by an average 33% (Kimball, 1983). Thus if it were possible to improve the efficiency with which CO2 competes with 0 2 at the active site of the Rubisco enzyme, the rate of photosynthesis would increase relative to the rate of photorespiration, with subsequent increases in crop yield (Ellis & Gatenby, 1984).

Evolution has produced a means of reducing photorespiration in some species, but always by means of mechanisms that increase the concentration of carbon dioxide around the active site of the enzyme, such as by pumping CO2 across membranes which occurs in

unicellular algae and cyanobacteria (Badger et al., 1980; Spalding et al., 1983), and by the

C4 type o f photosynthesis found in some families of higher plants (Edwards & Walker, 1983). However the observed natural variation of the kinetic properties of the Rubisco enzyme from different sources indicates that modifications improving the efficiency of carboxylation have also occurred during evolution (Jordan & Ogren, 1981; 1983). It is because of this latter observation that Rubisco is a major target for genetic engineering to investigate whether it is possible to accelerate these evolutionary changes for the benefit of agriculture (Miziorko & Lorimer, 1983; Ellis & Gatenby, 1984).

Attemps to produce altered form of Rubisco by mutagenizing plant cells in order to prevent photorespiration have been unsuccessful so far (reviewed by Somerville & Ogren, 1982). In view of the difficulties in obtaining useful mutants, attempts have been made to express plant DNA in bacteria with the aim of assembling a functional Rubisco enzyme, that could provide the basis for mutagenic studies to alter the properties of the enzyme. These attempts have so far not succeeded in producing an active enzyme from DNA sequences

coding for the Rubisco subunits from higher plants (Bradley et al., 1986; Gatenby et al.,

cyanobacterial sequences. The synthesis and assembly o f Rubisco subunits in Escherichia coli (E.coli) is reviewed in detail in Section 1.4.

The synthesis and assembly of the hexadecameric Rubisco enzyme in higher plants involves light and the coordinated interaction of two distinct genetic systems present in the chloroplast and the nucleus. The understanding of this clearly complex process has been of continuing interest to scientists and the next sections review and discuss some of the research which has contributed to our understanding of the synthesis and assembly o f the Rubisco enzyme (Section 1.2.3 and Section 1.2.4), the regulation of gene expression (Section 1.2.5) and the structural (Section 1.2.1) and catalytic properties (Section 1.2.2) of the enzyme. The failure to assemble an active higher plant Rubisco enzyme from its subunits synthesized in

E .co li, coupled with the observations made by Barraclough & Ellis (1980) have led to the proposal that another protein may be required for the assembly of Rubisco in plants. The discovery and the properties of this protein, called the Rubisco large subunit binding protein, are reviewed in Section 1.3.

The aim of this review is to describe and discuss observations that have led to the hypothesis that the co-synthesis of the Rubisco large subunit binding protein in the same

E.coli cell that synthesizes the Rubisco subunits may be a way of rescuing the assembly of a functional enzyme, and thus allowing mutagenic studies to proceed.