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How recruiting a novel subunit enabled Rubisco, the carboxylase of photosynthesis, to evolve higher specificity

X-ray crystallography was used to re-trace the evolution of a novel subunit in the carboxylase Rubisco. This subunit enabled Rubisco to evolve higher specificities for CO2 over O2.

The appearance of oxygen in Earth s atmosphere presented a major challenge for organisms at the time. Organisms, enzymes and metabolisms that arose billions of years ago in anaerobic environments suddenly had to adapt to its presence. Among the organisms negatively impacted by oxygen were those that used Rubisco, the carboxylase of the Calvin cycle, for autotrophic growth. Rubisco also reacts with O2 instead of CO2, thereby producing an undesired compound that needs to be metabolised in an energy- and carbon-releasing reaction (Figure 32a).

From studying modern forms of Rubisco that evolved in aerobic environments, it was found that one of the ways evolution dealt with Rubisco s confused substrate scope was to improve its specificity for CO2 over O2. The molecular determinants of this increased CO2 specificity remained largely unknown, even though they are of great interest to researchers aiming to improve photosynthesis. The sole hint stemmed from the fact that Rubisco with increased CO2 specificity recruited a novel protein subunit of unknown function, the so-called small subunit (SSU) (Figure 32b). This SSU was suspected to be involved in increasing CO2 specificity, however, the true reason for its emergence remained difficult to determine because

it already evolved billions of years ago and is strictly essential in proteins that exist today [1].

To re-trace and understand this event, a statistical algorithm called ancestral sequence reconstruction was used to recreate forms of Rubisco that existed billions of years ago [2], shortly before and shortly after the SSU evolved. The ancestral Rubisco that existed shortly before the emergence of the SSU could not yet interact with it and was not specific. Conversely, the ancestral Rubisco that existed shortly after the SSU s emergence was already dependent on it and had increased specificity. Importantly, the historical substitutions separating these two ancestral Rubiscos must be responsible for creating the interaction with the SSU and the dependence thereon.

Analysis of the ancestral Rubisco s crystal structures at beamline ID23-2 yielded important insights into where in the protein the historical substitutions occurred and which residues were important for the Rubisco-SSU interaction (Figure 32c). Based on these insights, an intermediate Rubisco was created that was able to bind the SSU but did not yet depend on it. Using these variants, it was shown that immediately upon its recruitment, the SSU improved Rubisco s catalysis and opened evolutionary paths that led to increased CO2 specificity (Figure 32d). Notably, the SSU alone was not the cause of drastic changes in specificity. Instead, it modulated the effects of further substitutions. Residues without effect in isolation are suddenly influential in SSU presence. The SSU seemingly teleports Rubisco to a new functional sequence space.

A similar effect was responsible for the SSU addiction. Accumulation of substitutions that were harmless in the

Fig. 32: a) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) can catalyse both a carboxylation and an oxygenation reaction. b) Rubiscos found in aerobic environments evolved from a dimeric assembly of large subunits (L2) to a hexadecameric hetero-

complex consisting of eight large and eight small subunits (L8S8). PDB: protein data bank. c) Analysis of Rubisco s crystal structure yielded important insights into which historical amino acid substitutions occurred in proximity to the small subunit (SSU) binding

site, when the SSU first evolved. d) Presence of the SSU increases Rubisco s specificity for CO2 over O2, enabling better discrimination between the two gases. e) Rubisco evolves to depend on the SSU for solubility and forms insoluble fibres when the SSU is not present.