Corlett, T. | Search bioRxiv

bioRxiv2026

Nucleotide-binding motifs nucleated folding of the first enzymes

The earliest stages of protein evolution remain a mystery: the nature of the first protein forms, their roles in emergent biological systems, and the forces that shaped them are largely unknown. Here, we combine insights from metabolic modelling, the organization of protein structure space, and protein folding mechanisms to probe the emergence of two ubiquitous cofactor- binding folds: Rossmanns and P-loop NTPases. While both folds are essential for contemporary life, we show that Rossmanns catalyze reactions deeper within the metabolic core and are more central in structure space than P-loop NTPases. Folding mechanism analysis further reveals that, whereas P-loop NTPases may require non-local interactions to fold, Rossmann folding can be nucleated by a structural module at the heart of the fold that contains a nucleotide-binding motif. Because this motif also directly mediates biochemical activity, this result suggests how early proteins may have compactly satisfied both folding and biochemical activity. Our results imply that folding constraints favored early enzymatic forms with compact binding motifs and modest catalytic roles. We conclude that the early emergence of the Rossmann fold reflects the chemical and physical constraints of protein folding, explaining both its profound antiquity and sustained longevity.

biochemistry↗

bioRxiv2025

The History of Enzyme Evolution Embedded in Metabolism

Whereas phylogenetic reconstructions are a primary record of protein evolution, it is unknown whether the deep history of enzymes are encoded at higher levels of biological organization. Here, we demonstrate that the emergence and reuse history of enzymatic folds is embedded within the web of metabolite-cofactor-enzyme interdependencies that comprise biosphere-scale metabolic reaction networks. Using a simple network analysis approach, we reconstruct the relative ordering of enzymatic fold emergence and, where possible, the first reaction(s) that each enzymatic fold catalyzed. We find that a large majority of enzymatic folds were sufficient as independent additions to open new avenues for metabolic growth. The resulting network-based histories are broadly concordant with enzyme phyletic distribution in prokaryotes, a proxy for enzyme age. Our results suggest that the earliest enzyme-mediated metabolisms were enriched for /{beta} proteins, likely due to their strong association with cofactor utilization, and that -proteins preferentially emerge at later stages. The cradle-loop barrel, a member of the small {beta}-barrel metafold, is predicted to be the founding {beta}-fold, in agreement with analyses of ribosome structure. An examination of how the protein universe responded to the biological production of molecular oxygen reveals that the adaptation of existing enzymatic folds, not novel fold emergence, was the primary driver of metabolic evolution. This work presents a self-consistent model of metabolic and enzyme evolution, key progress towards integrating diverse perspectives into a unified history of protein evolution. Significance StatementEnzyme emergence is an ongoing process that began [~]4 billion years ago. Here, we show that the modern biosphere-scale network of metabolic reactions and enzymes is an archive of enzyme history independent from, but concordant with, phylogenetics. Based on this record, we predict the order of enzyme emergence from before the last universal common ancestor up until the biological production and metabolic utilization of molecular oxygen. We find that while /{beta} proteins dominated primitive enzyme-mediated metabolism, other folds -- including the cradle-loop barrel, which is a member of the small {beta}-barrel metafold -- were likely important early contributors. This study represents key progress towards building an internally consistent, joint history of metabolic reactions and enzymes.

bioinformatics↗