Structural studies of different form I Rubiscos using molecular dynamics simulations

Abstract

Photosynthesis is the process by which autotrophic photosynthetic organisms utilise light energy to assimilate CO2 into biomass, releasing O2 into the atmosphere as a by-product. Even though photosynthetic reactions were crucial in the “Great Oxygenation Event” of our atmosphere 2.4 billion years ago, these greatly limit crop yields. Hence, increasing the photosynthetic efficiency of light conversion into biomass has become a crucial practice to feed the increasing global population. Rubisco is a fundamental enzyme in the carbon reactions of photosynthesis, which fixates atmospheric carbon dioxide into biomass. However, due to its slow turnover (3 molecules of CO2 fixed per second) and inhibition of carboxylation reactions by oxygenation, Rubisco is a major bottleneck of carbon fixation in photosynthesis. Rubisco form I from higher plants is the most abundant form of Rubisco on earth. It is a complex enzyme consisting of 8 large and 8 small subunits, which forms a hexadecameric structure with a mass of 550 kDa in higher plants. Due to Rubisco’s multimeric nature, targeted mutagenesis experiments to investigate more efficient catalysts in higher plants is extremely challenging. Furthermore, its eight active sites are located in the interface of the large subunits, a feature which further complicates the understanding of the events occurring in the active sites responsible for Rubisco’s catalytic inefficiencies. While molecular dynamics (MD) simulations can be used to investigate these inefficiencies, previous studies are limited by a 50 ns time frame, thereby lacking the ability to adequately capture the underlying structural dynamics. For the first time, this thesis presents 17 long MD simulations, ranging from 500-1500 ns, using 13 different structures of Rubisco form I from three distinct organisms (Synechoccocus, spinach and Chlamydomonas). It provides evidence of the suitability of this technique in inspecting the impact of different mutants of Rubisco on the RuBP substrate’s binding affinity. For this purpose, results were compared to existing experimental data of mutant forms of Rubisco. The essays hereby reported demonstrate that, after long MD simulations of Rubisco, the resulting binding affinity ranking of the substrate to different mutants is consistent with previous experimental work. Moreover, the simulations reveal an allosteric behaviour of the substrate binding between the eight active sites of Rubisco, and verify the influence of Rubisco’s structural elements on the binding affinity of its substrate.