Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco

Abstract

Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO 2 fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit ␣͞␤-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO 2͞O2 specificity, despite the fact that land-plant Rubisco has greater specificity than the Chlamydomonas enzyme. Because the residues are close to a variable loop between ␤-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When largesubunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO 2͞O2 specificity to the normal level. Further substitution with the shorter ␤A-␤B loop of the spinach small subunit caused a 12-17% increase in specificity. The enhanced CO 2͞O2 specificity of the mutant enzyme is lower than that of the spinach enzyme, but the carboxylation and oxygenation kinetic constants are nearly indistinguishable from those of spinach and substantially different from those of Chlamydomonas Rubisco. Thus, this interface between large and small subunits, far from the active site, contributes significantly to the differences in catalytic properties between algal and land-plant Rubisco enzymes.catalysis ͉ Chlamydomonas ͉ chloroplast ͉ photosynthesis ͉ ribulosebisphosphate carboxylase͞oxygenase C O 2 and O 2 compete at the active site of ribulose-1,5-bisphosphate (RuBP) carboxylase͞oxygenase [Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), Enzyme Commission 4.1.1.39] for either the carboxylation or oxygenation of RuBP (reviewed in refs. 1-3). Whereas carboxylation is responsible for the accumulation of carbon in the biosphere, oxygenation is a nonessential reaction that leads to the loss of fixed carbon via the photorespiratory pathway. The ratio of the catalytic efficiencies (V max ͞K m ) of carboxylation (V c ͞K c ) and oxygenation (V o ͞K o ) defines the CO 2 ͞O 2 -specificity kinetic constant ⍀ (4), which is determined by the differential stabilization of the carboxylation and oxygenation transition states for the rate-limiting partial reactions (5). However, net CO 2 fixation is determined by the difference between the velocities of carboxylation and oxygenation at the CO 2 and O 2 concentrations that occur in vivo (4, 6). Because of its pivotal role in catalyzing the rate-limiting step of photosynthesis, genetic engineering of Rubisco aimed at increasing net CO 2 fixa...

Isoleucine 309 acts as a C 4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

“…3) (24,29,30) contrasts with the recent success in shaping rice rubisco toward C 4 -like catalysis using heterologous S-subunits from C 4 sorghum (16). Similarly, structural changes to the S-subunit have improved Chlamydomonas rubisco catalysis (17). As highlighted recently (20), differences in rubisco S-subunit sequence also may account for the catalytic deviation of Flaveria palmeri rubisco, whose L-subunit sequence matches that of tob flo-bid (coding 309 Ile) but shows C 3 catalysis.…”

“…The catalytic core of L 8 S 8 rubisco comprises four 52-kDa large (L)-subunit pairs which are capped by two sets of 15-kDa small (S)-subunit tetramers that provide structural stability and influence catalysis (16,17). Although supplementing rice rubisco with Ssubunits from the C 4 plant sorghum was found to improve V C of the heterologous L 8 S 8 enzyme (16), crosses between C 3 and C 4 Flaveria and Atriplex species showed C 4 catalysis to be maternally inherited (18,19) and hence defined by the chloroplast-encoded L-subunit gene (rbcL).…”

Stability-activity tradeoffs constrain the adaptive evolution of RubisCO

“…Whereas adaptive mutations 10-20 Å from active sites have occasionally been identified in other enzymes (31), in RubisCO, these form the majority of positively selected sites that distinguish C 3 and C 4 species. Experiments with RubisCO from the green alga Chlamydomonas reinhardii previously implicated the interfaces between large and small subunits in the modulation of catalytic rates (32). The analysis here increases the number of known functionally significant intersubunit sites (Table S2) and demonstrates a link with the C 3 -C 4 transitions in flowering plants.…”

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits

“…Directed mutagenesis of Rubisco from the cyanobacterium Synechococcus expressed in Escherichia coli and directed mutagenesis and genetic selection in vivo in the green alga Chlamydomonas reinhardtii have identified several residues of the small subunit that can influence ⍀ (17-21). When the longer loop between ␤-strands A and B of the Chlamydomonas small subunit was replaced with the shorter loop of Synechococcus, a decrease in ⍀ was observed (15,22 …”

“…However, some plant and algal species contain CO 2 -concentrating mechanisms (CCMs) (reviewed in Refs. 10 -14), which make it difficult to tell whether any Rubisco enzyme in nature is better than any other with respect to the CO 2 and O 2 concentrations (and temperatures) that the enzyme encounters in vivo (1,15). Nonetheless, various mutant substitutions have been identified that can influence ⍀ (reviewed in Refs.…”