Role of Half-of-Sites Reactivity and Inter-Subunit Communications in DAHP Synthase Catalysis and Regulation

alpha-Carboxyketose synthases, including 3-deoxy-D- arabinoheptulosonate 7-phosphate synthase (DAHPS), are longstanding targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-ofdimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4phosphate (E4P). The N-terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all alpha-carboxyketose synthase families and a regulatory role in some members.

Automated summary

This study reveals the structural and dynamic basis for inter-subunit communication in Escherichia coli DAHPS(Phe) using X-ray crystallography and HDX. The enzyme exhibits pseudo-twofold symmetry, and key parts of the communication network include the N-terminal peptide and the active site loop that binds the substrate. Inter-subunit communication has a catalytic role in all alpha-carboxyketose synthase families and a regulatory role in some members.