Designing Stacked Assembly of Type III Rubisco for CO₂ Fixation with Higher Efficiency

Abstract

The slow catalytic rate of the carboxylation enzyme D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major barrier to increasing the rate of carbon assimilation from the atmosphere into the biosphere. It is of great importance to establish a method to improve the carboxylation efficiency of Rubisco. Inspired by the assembly of Rubisco in carboxysomes, herein, we presented a rational protein engineering approach for the construction of one-dimensional (1D) protein arrays of type III Rubisco through designed π−π stacking interactions by using crystal structural information as a guide. In aqueous solutions, the dimensions of these 1D protein arrays collectively span nearly the entire nano-and micrometer scale (200 nm to 5.0 μm) by adjusting protein and NaCl concentrations. As a result, the stacked Rubisco assemblies increase by 40% in the carboxylase activity, while their turnover number (k cat ) is around twofold larger than that of wild-type III Rubisco. Notably, upon heat treatment at temperature up to 75 °C for 30 min, most of the assembled nanostructures and the enzyme activity are retained. More importantly, the initial relative activity of stacked assemblies retained 91% after 10 cycles of reuse. This work provides a simple, effective solution for the improvement of the CO 2 carboxylation efficiency of Rubisco.

Structural plasticity enables evolution and innovation of RuBisCO assemblies

“…Our results demonstrate how a small number of residues-only seven mutations-can enable an increase in oligomeric state, providing insight into the requisite degree of plasticity necessary for innovation of larger oligomeric states. Recent studies have demonstrated how few mutations can be introduced to proteins, resulting in radical increases in oligomerization that are more accurately described as non-native protein fibrils (27)(28)(29). In comparison, our study focuses on understanding the structural basis underlying transitions between oligomeric states found in nature, as our mutational engineering of the 2-to-6 enzyme recapitulates the evolutionary trajectory taken by form II RuBisCOs when assembling hexamers from dimers.…”

Role of carboxysomes in cyanobacterial CO2 assimilation: CO2 concentrating mechanisms and metabolon implications

“…Several recent approaches hold promise and indicate that novel strategies implementing modified carbon-fixing machinery could lead to substantial improvements over the status quo. For example, a recent study inspired by the packed arrangement of RuBisCO within carboxysomes generated synthetic stacked assemblies of RuBisCO that exhibited enhanced catalytic activity and stability (Zeng et al, 2022). Incorporation of RuBisCO activase within carboxysomes is another strategy to reduce the inactivation of RuBisCO and enhance carbon-fixation (Chen et al, 2021).…”