Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes the first step in net photosynthetic CO2 assimilation and photorespiratory carbon oxidation. The enzyme is notoriously inefficient as a catalyst for the carboxylation of RuBP and is subject to competitive inhibition by O2, inactivation by loss of carbamylation, and dead-end inhibition by RuBP. These inadequacies make Rubisco rate limiting for photosynthesis and an obvious target for increasing agricultural productivity. Resolution of X-ray crystal structures and detailed analysis of divergent, mutant, and hybrid enzymes have increased our insight into the structure/function relationships of Rubisco. The interactions and associations relatively far from the Rubisco active site, including regulatory interactions with Rubisco activase, may present new approaches and strategies for understanding and ultimately improving this complex enzyme.

A series of non-photoautotrophic mutants of Chlamydomonas reinhardii was isolated by replica-plating mutagenized cells which had been grown in the dark. Many of these acetate-requiring mutants are photosensitive, showing poor growth on acetate medium in the light, but normal growth in the dark. Biochemical characterization showed that the photosensitive mutants all had specific lesions in photosynthesis or photosynthetic pigment accumulation. The acetate-requiring mutants which were not photosensitive were all able to fix CO2. Among the light-sensitive mutants are 15 which show uniparental inheritance. These include six with specific lesions in photosystem II and one with an altered

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A mendelian mutant of the unicellular green alp Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-i), was selected on the basis of a high C02 requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric C02 concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by 02. In contrast to a CO2 compensation concentration of near zero in wild type at all 02 concentrations examined, the mutant exhibited a high, 02-stimulated C02 compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of '4CO2. At

The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 Å resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between ␤ strands A and B of the small subunit (␤A-␤B loop), which is longer by six amino acid residues than the corresponding region in Rubisco from Spinacia. Mutations of residues in the ␤A-␤B loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonas structure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.Ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39; Rubisco) 1 catalyzes the addition of CO 2 to RuBP (reviewed in Refs. 1-3). This reaction initiates photosynthetic carbon assimilation via the Calvin cycle and results in the net gain of carbon from atmospheric CO 2 into the biosphere. The carboxylation reaction of Rubisco is the major source of carbon needed for life. A competing reaction in which O 2 is added to RuBP results ultimately in a net loss of CO 2 , a process known as photorespiration. Because the reaction catalyzed by Rubisco is unique to photosynthetic CO 2 fixation and nearly all carbohydrate production is dependent on it, the oxygenase reaction qualifies as the most important limiting factor of photosynthetic yield.All Rubisco enzymes have oxygenase activity. This is true also for strictly anaerobic microbes, including some anaerobic archaebacteria (reviewed in Ref. 1). Thus, whereas the ratio of carboxylation to oxygenation at any specified concentrations of CO 2 and O 2 depends on the catalytic efficiency (k cat /K m ) of carboxylation relative to that of oxygenation, referred to as the CO 2 /O 2 specificity factor, net CO 2 fixation is determined by the difference between the rates of carboxylation and oxygenation (4, 5). The kinetic constants that determine carboxylation and oxygenation vary considerably among Rubisco enzymes from different species (6).Based on their quaternary structures, different forms of Rubisco enzymes can be distinguished (reviewed in Refs. 1 and 2). In cyanobacteria and plants with chloroplasts, such as red and brown algae and green plants, a hexadecameric (L8S8) form is found, consisting of eight 55-kDa L subunits and eight 15-kDa S subunits. A dimeric (L2) enzyme, similar in structure to the L subunits of the hexadecameric enzymes, is restricted to some bacteria and alveolates. More recently, a third form was discovered in the thermophilic archaebacterium Thermococcus kodakaraensis that comprises 10 L sub...