Functional conservation with mechanistic drift: AMP activation in the evolution of archaeal sugar kinases

Although allosteric regulation has been pointed out as one of the cornerstones of biological function, it has been a scarcely studied phenomenon in Archaea carbohydrate metabolism. Given its central role in metabolism, we experimentally investigated how allosteric regulation and its underlying kinetic mechanism evolved along evolutionary pathways within the archaeal ADP-dependent kinase family. Using ancestral sequence reconstruction, we resurrected key ancestors of this family and show that AMP regulation is an ancestral feature retained exclusively in lineages encoding bifunctional ADP-dependent phosphofructokinase (PFK)/glucokinase (GK) enzymes, which are restricted to methanogenic organisms, whereas it is lost in lineage-specific PFK enzymes. Notably, although AMP-dependent allosteric regulation is conserved among bifunctional ADP-PFK/GK enzymes, the kinetic mechanisms underlying activation are not. Instead, we observed a diversity of activation mechanisms (increased affinity for substrates, enhanced catalytic efficiency, or a combination of both), distributed along a 2-billion-year evolutionary trajectory, and that persists across different temperatures studied, both in extant and ancestral enzymes. These results highlight that the structural scaffold of this protein family is evolutionarily robust, preserving function while allowing substantial diversification of the underlying activation mechanisms under sequence variation. Based on these findings, we propose the concept of mechanistic drift, in which evolutionary pressures primarily act on adaptive functional traits that confer an adaptive advantage, rather than on the specific molecular mechanisms by which they are achieved. This framework has broad implications for macromolecular evolution, illustrating how long-term functional conservation can coexist with extensive physicochemical mechanistic diversity.