An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors

Abstract

Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose’s ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint—geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif.

Abstract

The widely distributed Rossmann-fold enzymes share a highly conserved geometry of their ribose binding motif; this geometry is very rarely found in other folds and represents a relic of a common ancestral enzyme.

Author Summary

Common descent is the hallmark of Darwinian evolution. Homology of biological traits, and particularly of protein sequences and structures, serves as an indication for divergence from a common ancestor and a means of assigning phylogenetic relationships. However, because of shared functional demands and chemical-physical constraints, proteins that evolved independently of one another often converge on very similar molecular traits, including structure and sequence. We tested the widely accepted hypothesis of common ancestry of several major enzyme classes, comprising hundreds of different families and using different cofactors and catalytic chemistries. Although they share the same overall architecture—the Rossmann fold—these enzymes show no significant sequence homology across different classes. We describe an analysis based on the omnipresence of a single residue across these classes: an acidic aspartate or glutamate residue that binds ribose, the common denominator of the different cofactors used by these enzymes. We show that Rossmann enzymes possess a unique interaction geometry that represents a fingerprint of common ancestry rather than an outcome of molecular constraint. We thus provide the first systematic test of divergence versus convergence of a highly abundant protein motif and assign common descent in one of the most ancient and functionally diverse protein folds.