Functional analysis of Rossmann-like domains reveals convergent evolution of topology and reaction pathways

Rossmann folds are ancient, frequently diverged domains found in many biological reaction pathways where they have adapted for different functions. Consequently, discernment and classification of their homologous relations and function can be complicated. We define a minimal Rossmann-like structure motif (RLM) that corresponds for the common core of known Rossmann domains and use this motif to identify all RLM domains in the Protein Data Bank (PDB), thus finding they constitute about 20% of all known 3D structures. The Evolutionary Classification of protein structure Domains (ECOD) classifies RLM domains in a number of groups that lack evidence for homology (X-groups), which suggests that they could have evolved independently multiple times. Closely related, homologous RLM enzyme families can diverge to bind different ligands using similar binding sites and to catalyze different reactions. Conversely, non-homologous RLM domains can converge to catalyze the same reactions or to bind the same ligand with alternate binding modes. We discuss a special case of such convergent evolution that is relevant to the polypharmacology paradigm, wherein the same drug (methotrexate) binds to multiple non-homologous RLM drug targets with different topologies. Finally, assigning proteins with RLM domain to the Enzyme Commission classification suggest that RLM enzymes function mainly in metabolism (and comprise 38% of reference metabolic pathways) and are overrepresented in extant pathways that represent ancient biosynthetic routes such as nucleotide metabolism, energy metabolism, and metabolism of amino acids. In fact, RLM enzymes take part in five out of eight enzymatic reactions of the Wood-Ljungdahl metabolic pathway thought to be used by the last universal common ancestor (LUCA). The prevalence of RLM domains in this ancient metabolism might explain their wide distribution among enzymes. Author summary Protein-ligand interactions are crucial to understanding molecular-level mechanisms of cell functions. A ligand-binding site frequently acts as a protein's functional center, where its structural scaffold orients molecules in the correct conformation for proper function such as catalysis. These binding or active sites are usually conserved within protein families and can be used to define homologous proteins. However, even homologs may have diverged to functions requiring different active sites. Here we study the structural features and evolution of active sites of proteins containing the Rossmann-like motif (RLM). RLM proteins are numerous in nature and they constitute about 20% of all known 3D structures. Taking into account the prevalence of RLMs in proteins, analysis of their ligands provides general insights into enzyme evolution as well as proposes possible domain-based strategies for drug design in light of polypharmacology. Our analysis shows that RLM enzymes function predominantly in metabolism-these proteins cover 38% of reference metabolic pathways and are involved in numerous metabolic diseases. Being one of the most ancient folds, RLM enzymes could be traced to ancient metabolic pathways used by LUCA, which are still in use in some bacteria today.