Structure of an ancestral ADP-dependent kinase with fructose-6P reveals key residues for binding, catalysis, and ligand-induced conformational changes

Abstract

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-PFKs), specific glucokinases (ADP-GKs), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, none exhibits fructose-6-phosphate (F6P) at the active site. Using an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with F6P at its active site. Key residues for sugar binding and catalysis were identified by alanine scanning, D36 being a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding because 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, semiclosed, and closed).

Catalytic mechanism for Renilla-type luciferases

“…High protein stability is known to facilitate crystallization [27][28][29] , and it has been demonstrated that the structures of ancestral enzymes can be used as molecular scaffolds to unravel the structures and catalytic mechanisms of extant enzymes that are difficult to crystallize. [30][31][32][33][34][35] We previously used ancestral sequence reconstruction to obtain a stable ancestral enzyme Anc HLD-RLuc that was reconstructed from the catalytically distinct but structurally related HLDs and Renilla luciferase. 9 This ancestor enzyme turned out to have dual functions, with dehalogenase activity comparable to that of contemporary HLDs as well as promiscuous luciferase activity significantly lower than that of the stabilized RLuc8.…”