# Unraveling the regioselectivity of Ophiostoma piceae sterol esterase as a case study for lipases with wide acyl-binding tunnel entrances

## TL;DR

This study explores how the structure of a wide tunnel in a fungal enzyme determines its preference for breaking specific fat bonds, offering insights into enzyme design for lipid synthesis.

## Contribution

The study reveals how a wide acyl-binding tunnel entrance in OPE leads to sn-1(3) regioselectivity, expanding understanding of enzyme structure-function relationships.

## Key findings

- OPE shows 85.8% sn-1(3) regioselectivity against TOG, contrasting with Candida antarctica lipase A's 79.8% sn-2 selectivity.
- Molecular dynamics simulations show the wide tunnel entrance stabilizes sn-1(3) binding without disrupting catalytic interactions.
- Tunnel architecture directly dictates the regioselectivity of lipases with acyl-binding tunnels.

## Abstract

Lipase regioselectivity (sn-1(3) vs sn-2) is crucial for synthesizing structured lipids, but the structural basis for contrasting regioselectivities in lipases with acyl-binding tunnel remains incomplete. While the correlation between a narrow tunnel entrance and sn-2 regioselectivity has been previously established, the regioselectivity of Ophiostoma piceae sterol esterase (OPE), a lipolytic enzyme with a uniquely wide tunnel entrance, remained unclear. In this study, the chiral-phase resolution of oleic species confirmed that OPE exhibits a notable sn-1(3) regioselectivity (85.8%) against trioleoylglycerol (TOG), in contrast to Candida antarctica lipase A, which possesses narrow tunnel entrance (sn-2 regioselectivity: 79.8%). Molecular dynamics simulation showed that the wide tunnel entrance of OPE facilitates stable interaction of the scissile ester group within the catalytic center only when adopting sn-1(3) binding mode; the additional C1(3)–C2 glycerol backbone linkage within TAG allows non-scissile chains to stably reside within the tunnel entrance without disrupting the interactions at the catalytic center. Overall, the results indicated that the tunnel architecture directly dictates the sn-1(3) regioselectivity of OPE, providing structural insights into the tunnel morphology–regioselectivity relationship for lipases with acyl-binding tunnel.