Metabolites form a globally connected chemical network across protein families

Metabolites are generally viewed as substrates, products, cofactors, or regulators of individual proteins, whereas metabolites recurring across many protein families are often regarded as promiscuous binders. Here, we analyzed 989,058 BioLiP2 protein-ligand binding sites and assigned 929,546 sites to ECOD v295 homologous groups to quantify ligand specificity, cross-fold scatter, structural breadth, and metabolite-mediated connectivity across protein-family space. Many ancient metabolites preferentially occupied cognate structural groups, demonstrating that broad evolutionary reuse can coexist with local structural discrimination. After excluding elemental metals, BioLiP potential-artifact/dual-use ligands, and metabolites containing fewer than six heavy atoms, 32 ancient metabolites occupied a mean of 185.38 ECOD F-groups per metabolite, compared with 6.32 F-groups for 2,540 mapped filtered non-ancient metabolites-- a 29.35-fold enrichment (bootstrap 95% CI, 18.66-43.46). The complete 40-ancient-metabolite network connected all 6,798 associated F-groups into a single giant connected component (GCC). Even after stringent filtering, all 3,135 ancient-metabolite-associated F-groups remained in one GCC. Degree-preserving configuration-model randomizations and maximum-degree capping showed that this connectivity follows from the broad, recurrent distribution of metabolite binding rather than dependence on a few extreme hubs or a specialized higher-order topology. Differences between ancient and filtered non-ancient networks were not explained by metabolite size, whereas generic crystallization additives preferentially occupied smaller pockets. These results indicate that a limited ancient chemical repertoire established a globally connected protein- family architecture that subsequent metabolite diversification expanded while preserving its basic organization. SignificanceMetabolites are conventionally viewed as substrates, products, cofactors, or regulators acting on individual proteins. Global examination of experimentally observed metabolite-protein interactions reveals a broader organizing principle. Ancient metabolites combine local binding discrimination with extraordinary reuse across protein families, such that only 40 metabolites generate an almost completely connected network spanning thousands of ECOD (evolutionary classification of domains) protein families. The much larger non-ancient metabolite repertoire expands the protein-family space covered by this network, while preserving near-global connectivity. Thus, metabolite diversification appears to have elaborated, rather than created, a chemically connected protein architecture established early in evolution, suggesting that overlapping metabolite-binding repertoires could coordinate proteins, pathways, and cellular processes.