Evolutionary insights into the structural basis of allosteric regulation and K⁺-independent activity in pyruvate kinases from methanogenic archaea

Pyruvate kinase (PK) catalyzes the final step of glycolysis, converting phosphoenolpyruvate (PEP) and ADP into pyruvate and ATP. Although PKs are homologous across all domains of life, their allosteric regulation shows remarkable diversity. In eukaryotes and many bacteria, fructose-1,6-bisphosphate acts as an activator, whereas in other bacteria and in methanogenic archaea, AMP fulfills this role. Moreover, PKs display an almost dichotomous activity dependence on potassium ions: enzymes with a glutamate residue at the active site are K+-dependent, while those with a lysine at the same position function independently of K⁺.

To date, no structural basis has been established for the evolution of allosteric regulation in PKs from methanogenic archaea, nor has the structural basis for K⁺-independent activity been elucidated. To address this gap, we employed ancestral sequence reconstruction to investigate the evolutionary origin of AMP specificity at the allosteric site of PK enzymes in methanogenic archaea. A phylogenetic tree was generated from 411 PK sequences, from which the most probable sequences of the common ancestor of PKs across all three domains of life (AncCPK) and of the last common ancestor of the order Methanococcales (AncMcPK) were inferred.

We recombinantly expressed and purified both ancestral PKs. Functional assays demonstrated that AncCPK is K⁺-dependent, whereas AncMcPK is K⁺-independent. Cryo-EM structures of AncCPK, AncMcPK, and extant PKs from Methanococcus maripaludis and Methanocaldococcus jannaschii revealed AMP bound at the allosteric site in all cases, indicating that AMP regulation is an ancestral trait and suggesting that either a lysine residue or a positively charged ion is essential for AMP specificity. The high-resolution cryo-EM structure of AncMcPK (2.1 Å) showed that the lysine residue coordinates a water molecule, functionally replacing K⁺ in the stabilization of PEP and thereby providing a structural basis for K⁺-independent activity. Moreover, the cryo-EM structure of AncMcPK in the apo state revealed significant conformational changes upon AMP and PEP binding, including lid-domain closure and loop rearrangement within the allosteric site.

This work was supported by FONDECYT 1231263 (VG) and ANID Fellowship for PhD complementary funds 21230418 (SMM).