Protein Structuromics Reveals a Loop-Controlled Half-Open Active Pocket Conformation Throughout Fe(II)/α-ketoglutarate-Dependent Dioxygenase Catalytic Cycle.

The Fe(II)/α-ketoglutarate-dependent dioxygenase (αKGD) superfamily enables various C-H functionalization supported by their active pocket architectures composed of one or more loop elements. However, their conserved architectural features and broader functional roles across the catalytic cycle remain incompletely defined, although such loops have been implicated in substrate recognition or proton transfer in specific catalytic processes. Here, we applied protein structuromics analysis to the PF10014 family within the αKGD superfamily, which contains a single long active-pocket loop and therefore provides a tractable model for dissecting loop-associated structure-function relationships. This analysis identified a conserved structural motif termed the half-open active pocket, which is proposed as a hallmark feature of the αKGD superfamily. Furthermore, enhanced sampling simulations and mutagenesis experiments targeting isoleucine dioxygenase, a representative member of the PF10014 family, revealed the multifaceted role of the loop that constitutes the half-open active pocket architecture throughout the catalytic cycle, encompassing the modulation of half-open active pocket conformation, substrate recognition and anchoring, as well as participation in molecular transport. These findings provide a broader structure-function landscape in which the flexible loop within this family-conserved half-open active pocket serves as a key catalytic element with multiple functions.