FUT8 Catalysis Involves GDP-Fucose-Induced Loop Activation Promoting a Reaction at the S_N1‑S_N2 Frontier

Area of Science:

• Biochemistry and Structural Biology
• Enzymology and Catalysis
• Glycobiology and Glycoprotein Structure

Background:

• Alpha1,6-fucosyltransferase 8 (FUT8) is essential for core fucosylation of N-glycans, a vital modification for mammalian glycoprotein function.
• The precise structural and mechanistic details of FUT8 catalysis are not fully understood, limiting targeted therapeutic development.

Purpose of the Study:

• To elucidate the complete catalytic cycle of FUT8 using advanced computational simulations.
• To understand the structural dynamics and mechanistic pathways governing FUT8-mediated fucose transfer.

Main Methods:

• Integration of molecular dynamics, quantum mechanics/molecular mechanics (QM/MM), and metadynamics simulations.
• Analysis of enzyme-substrate interactions, conformational changes, and reaction coordinate topology.

Main Results:

• GDP-fucose binding induces conformational changes in flexible loops, stabilizing a catalytically active closed state.
• FUT8 employs a highly asynchronous SN2 inverting mechanism, proceeding through a late transition state in a single kinetic step.
• Transient ion pair formation observed, but no stable intermediate, despite a cationic transition state.

Conclusions:

• FUT8 utilizes structural rearrangements to achieve a unique catalytic process, distinct from other glycosyltransferases.
• The findings provide a detailed mechanistic understanding and a structural basis for designing FUT8 inhibitors.