ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFK), specific glucokinases (ADP-GK), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, no one exhibits fructose-6P at the active site. Employing an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with fructose-6P at its active site. Key residues for sugar-binding and catalysis were identified by alanine scanning, being D36 a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding since its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar-binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semi-closed and closed).
The Inner Interhelix Loop 4–5 of the Melibiose Permease fromEscherichia coliTakes Part in Conformational Changes after Sugar Binding
