UC Berkeley

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme—found in plants, algae, and cyanobacteria—forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I′) has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I′ rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I′′ rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I′′ rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution.Proteins form oligomeric complexes with other proteins, enabling a diversity of biological functions. The acquisition of protein-protein interactions between binding partners is generally considered to be the primary driver of oligomerization. However, ligand binding is similarly capable of driving higher-order assembly, though how these interactions contribute to and constrain the evolution of protein complex formation is less well understood. Here, we demonstrate how protein oligomerization can emerge via ligand-protein interactions within an enzyme family. Through phylogenetic sampling of a unique clade of rubisco, we discover a range of oligomeric responses to ligand binding across the phylogeny and identify the structural features driving an observed dynamic shift in oligomeric state. We further identify the critical role of a metal cofactor in governing higher-order assembly. Our results describe the acquisition of a ligand-protein interaction that mediates oligomerization in rubisco, illustrating a means by which stochastic binding and entrenchment of a ligand enables an increase in complexity.