Supplementary Materials Supplemental Data supp_284_44_30105__index. the remainder influence catalysis with or without some degree of connected structural instability (Y32A, Electronic43A, W73A, Odz3 L78A, P79A, and F81A). Y32A and E43A trigger reduces in CO2/O2 specificity. In line with the x-ray crystal framework of Rubisco, all except one (Glu-92) of the conserved residues are in touch with huge subunits and cluster close to the amino- or carboxyl-terminal ends of huge subunit -helix 8, that is a structural part of the /-barrel energetic site. Little subunit residues Glu-43 and Trp-73 determine a feasible structural connection between energetic site -helix 8 and the extremely variable little subunit loop between -strands A and B, that may also impact Rubisco order Avasimibe CO2/O2 specificity. In vegetation and green algae, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39)2 is made up of eight chloroplast-encoded large subunits and eight nuclear-encoded little subunits (known as L8S8 Rubisco) (reviewed in Refs. 1C3). The 55-kDa huge subunit contains a carboxyl-terminal /-barrel domain that, along with residues from the amino-terminal domain of a neighboring large subunit, forms the active site of the enzyme. Carboxylation of ribulose 1,5-bisphosphate (RuBP) initiates the rate-limiting step of photosynthetic CO2 fixation. However, O2 is mutually competitive with CO2, and oxygenation of RuBP is a nonessential side reaction that ultimately leads to the loss of CO2 in the photorespiratory pathway. Thus, net CO2 fixation is determined by the difference between the rates of carboxylation and oxygenation (4), which are ultimately determined by the values for CO2 ((referred to as L2 Rubisco) or the plant-like L8S8 Rubisco of expressed in (reviewed in Refs. 6 and 7). Numerous x-ray crystal structures are available to serve as a basis for such studies (2). However, as of yet, this information has not been exploited for designing a better Rubisco or for explaining the variation in kinetic constants observed for Rubisco enzymes from different species (8C11). Much less is known about the role of the 15-kDa small subunit in Rubisco function (reviewed in order Avasimibe Ref. 12). The small subunit is not in contact with any of the large subunit active site residues, but directed mutagenesis of prokaryotic and algal small subunits (13C15) or creation of hybrid enzymes comprised of large and small subunits from different species (16, 17) has indicated that the small subunit can also influence the value of . Because there is greater divergence between eukaryotic and prokaryotic small subunits than between large subunits, one wonders whether the small subunits may, in part, be responsible for the higher values of eukaryotic Rubisco enzymes (8). The loop between -strands A and B of the small subunit is the most variable structure among all Rubisco enzymes (12). It is comprised of 10 residues in prokaryotes and eukaryotic non-green algae, 22 residues in green plants, and 28 residues in green algae (see Fig. 1). The A-B loops of four small subunits, which reside at opposite ends of the octameric large subunit core, surround the opening of a solvent channel that passes through the holoenzyme (1, 2, 12). As a means for determining the significance of the longer loop in plant Rubisco, Bohnert and co-workers (28, 33) used transcription/translation and uptake into isolated pea chloroplasts to study the influence of engineered small subunits on the holoenzyme. This complex procedure was necessary order Avasimibe because eukaryotic holoenzymes cannot be expressed in (34) and because the small subunit is coded by a family of genes in plants that cannot be eliminated and replaced with engineered copies (35). When the longer A-B loop of plants was engineered into the small subunit, this cyanobacterial small subunit was then able to assemble with plant large subunits in the isolated chloroplasts (33). Thus, the A-B loop appeared to be an assembly domain (33), but detailed analysis of Rubisco catalysis was difficult to perform because of the low yield of holoenzyme in the isolated chloroplast system (28, 33). Open in a separate window FIGURE 1. Small subunit sequences aligned according to Rubisco x-ray crystal structures (18C23). The sequences are from (((((((((12). Individual residues previously investigated by mutagenesis are colored (13C15, 24C32). In the green alga gene family (comprised of linked and genes) of was deleted.