Protein Structuromics Reveals a Loop-Controlled Half-Open Active Pocket Conformation Throughout...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• The Fe(II)/α-ketoglutarate-dependent dioxygenase (αKGD) superfamily catalyzes crucial C-H functionalization reactions.
• Active pocket loops in αKGDs are implicated in substrate recognition and proton transfer but their broader roles are unclear.

Purpose of the Study:

• To investigate the structure-function relationships of active pocket loops in the αKGD superfamily.
• To elucidate the catalytic roles of a single long loop in the PF10014 family of αKGDs.

Main Methods:

• Protein structuromics analysis of the PF10014 family.
• Enhanced sampling simulations.
• Mutagenesis experiments on isoleucine dioxygenase.

Main Results:

• Identification of a conserved 'half-open active pocket' motif across the αKGD superfamily.
• The active pocket loop modulates pocket conformation, substrate binding, and molecular transport.
• The loop is essential throughout the enzyme's catalytic cycle.

Conclusions:

• The flexible loop within the conserved half-open active pocket is a key catalytic element with diverse functions.
• This study provides a comprehensive structure-function landscape for αKGD enzymes.
• Findings advance understanding of enzymatic C-H functionalization mechanisms.