# Catalytic Tunnel Dynamics Explain Functional Divergence of FAD2 Homologs Underlying the Oleic-to-Linoleic Ratio in Cornus wilsoniana

## TL;DR

Researchers studied how different versions of an enzyme in a woody oil tree affect the ratio of two fatty acids in its oil, using simulations to track how the enzyme's structure changes during binding.

## Contribution

The study introduces catalytic tunnel tracking to explain functional differences among FAD2 homologs in Cornus wilsoniana.

## Key findings

- One FAD2 version lacks a key protein segment and has a damaged binding pocket, likely rendering it nonfunctional.
- Two FAD2 versions form stable, open tunnels for efficient substrate positioning, while a third shows tunnel narrowing and substrate readjustments.
- CW02G01750 is identified as the leading structural candidate for regulating the oleic-to-linoleic acid ratio.

## Abstract

High-quality edible oils usually contain more oleic acid and less linoleic acid, but the molecular reasons for this balance remain unclear in the woody oil tree Cornus wilsoniana. We measured the main fatty acids in its fruit oil and then compared four closely related versions of an enzyme called fatty acid desaturase 2, which converts oleic acid into linoleic acid. One version lacked a large protein segment and showed a damaged binding pocket, suggesting it is unlikely to function properly. For the remaining three versions, we used molecular dynamics simulations together with catalytic tunnel tracking, a way to follow the passageway that guides the fatty acid to the reactive center, to watch how the protein shape changes during binding. Two versions formed a stable, more open access tunnel that kept the substrate well positioned, whereas the third showed higher internal flexibility and a tunnel that narrowed later in the simulation, forcing repeated substrate readjustments. These results prioritize CW02G01750 as the leading structural candidate among annotated C. wilsoniana FAD2 homologs and suggest that access-pathway dynamics may modulate substrate accommodation, providing a mechanistic hypothesis for future validation of O/L regulation.

The oleic-to-linoleic acid ratio (O/L) is a key determinant of oil quality, yet its molecular basis in Cornus wilsoniana remains unclear. Here, we combined fatty-acid profiling with molecular dynamics (MD) simulations and catalytic tunnel analysis to compare four annotated FAD2 homologs. Sequence alignment revealed a major variable segment at residues 160–185, including a small deletion in CW09G04700 and an extensive deletion in CW04G07690. Docking against oleic acid supported excluding CW04G07690 due to weak binding. Eighty-nanosecond MD simulations showed that CW02G01750 and CW09G27260 rapidly converged to stable conformational ensembles with lower core flexibility, whereas CW09G04700 exhibited higher internal mobility around residues 180–220. CAVER analysis further indicated increasingly accessible catalytic tunnels for CW02G01750 and CW09G27260 during simulation, while CW09G04700 displayed transient tunnel narrowing accompanied by ligand conformational readjustments. These results nominate CW02G01750 as a leading structural candidate among C. wilsoniana FAD2 homologs and highlight access-pathway dynamics as a mechanistic feature potentially contributing to O/L formation.