Retracing the evolution of rubisco oligomeric state

The majority of organic carbon on Earth has been sourced by nature’s primary carbon fixation enzyme: rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). As the most abundant enzyme on the planet, rubisco utilizes environmental carbon dioxide to generate two three-carbon intermediates, which, in photosynthesis, are used in the synthesis of complex sugars and are coupled to the release of molecular oxygen. However, rubisco’s overwhelming abundance belies the biochemical diversity found within its enzyme family. The aforementioned photosynthetic rubiscos represent only one form, form I, while bacteria, archaea, and other microorganisms comprise the remainder of rubisco forms: forms II, II/III, and III. Notably, the different rubisco forms contain different assemblies, or oligomeric states, with observed trends in each. The basic rubisco functional unit consists of a dimer of large subunits (2x RbcL - L2), arranged in a head-to-tail fashion. Form I enzymes are unique in their adoption of an additional small subunit (RbcS), where a central octameric core of four dimers is capped at both ends of the junctions between dimers, resulting in a hexadecameric assembly (L8S8). Form II enzymes are generally thought to be dimeric, though the recent discovery of two hexameric enzymes have identified purported outliers. One form II/III enzyme is known to increase in oligomeric state from a dimer to a decamer upon exposure to substrate, and form III enzymes are either dimeric or decameric. Considering the range of assemblies found in forms II, II/III, and III compared to the solely hexadecameric form I, two questions arise: how did dimeric assemblies evolve into a heterooligomer with an octameric core and what mechanisms drive variation in rubisco oligomerization? Form I rubiscos have historically been overrepresented in biochemical studies, and, as such, little is known about the remainder of the forms. Thus, diversity-driven structural studies are necessary to better understand the evolution of rubisco function and assembly, which may, in turn, guide future efforts to engineer the enzyme for improved carbon fixation. Here, I present studies into the evolution of oligomerization in newly discovered form I-adjacent, form II, and form II/III clades, retracing the evolutionary trajectories taken by the enzyme and identifying mechanisms underpinning the structural diversity observed in the present. Recent metagenomic experiments have identified rubisco sequences that cluster between the form I clade and all other forms on the phylogeny, suggesting the existence of extant enzymes representing evolutionary intermediates. To this end, we conducted biochemical and structural characterization of members of three clades, named form I’, form I’’, and form Iα. Through our analyses, we illuminate the acquisition of complexity from a dimer to an octamer lacking small subunits. Additional structural analyses revealed the relatively early acquisition of molecular features enabling small subunit acquisition in form I’’ enzymes, though these were lost in the form I’ clade. To further investigate patterns of oligomerization in rubisco, we conducted diversity-driven structural studies in sequences from both the form II and form II/III clades. Our studies of form II enzymes revealed an unprecedented level of structural plasticity, with observed interconversion, reversion, and innovation events that drive the diversity of oligomeric states observed at present. In form II/III enzymes, we identified two new patterns of oligomerization that were insensitive to substrate binding. Further investigation identified structural changes governing the previously identified oligomeric shift phenomenon, with my experiments demonstrating a critical function of a magnesium cofactor in maintenance of the form II/III decameric assembly. These findings further decrypt the evolutionary trajectory of rubisco, nature’s most prominent carbon fixation enzyme. As the scope and quantity of rubisco sequences continues to increase, future studies will further fill in gaps in phylogeny and structural knowledge and continue to retrace the evolution of oligomerization in this enzyme family. Studying rubisco oligomerization will not only be invaluable to understanding structure-function relationships, within rubisco and for other protein families at a broader scale, but also in guiding future protein engineering efforts to adapt the enzyme to an ever-evolving atmosphere