Diversity-driven biochemical survey reveals widespread dimerization throughout the rubisco superfamily

Rubisco is the entry point of nearly all organic carbon into the biosphere and is present in all domains of life. Despite its global importance, biochemical studies of this enzyme superfamily have been limited to a relatively narrow set of subclades. Recent advances in metagenomics have dramatically reshaped our understanding of both microbial and rubisco diversity; however, biochemical characterization of these sequences has not kept pace with the exponential growth in sequence data. To better survey the functional and structural diversity of rubisco, we systematically sample and synthesize a library of diverse rubisco sequences with an emphasis on clades that are sparsely represented in the biochemical literature. Our updated phylogenetic analysis reveals that many deep‑branching rubiscos assemble as dimers, supporting a dimeric origin for the superfamily - in contrast to the ecologically dominant hexadecameric form I. Additionally, we discover and structurally characterize an unusually large catalytic subunit among characterized rubiscos, originating from a early-branching subclade with secondary structural elements not present in canonical rubisco architectures.