# AlphaFold3-guided optimization of a photoactivatable endonuclease for top-down genome engineering

## TL;DR

Using AlphaFold3, researchers improved a light-controlled enzyme for precise genome editing, enhancing its DNA interaction and cleavage activity.

## Contribution

The study introduces a redesigned photoactivatable endonuclease with improved DNA binding and cleavage via AlphaFold3-guided structural modeling.

## Key findings

- AlphaFold3 modeling identified a split-site variant of MagMboI with increased DNA interface area and complex stability.
- The redesigned MagMboI-plus variant showed enhanced DNA cleavage activity in yeast upon blue light activation.
- MagMboI-plus induced more pronounced genomic rearrangements compared to the original construct.

## Abstract

Recent advances in protein structure prediction by artificial intelligence have enabled the rational design of engineered enzymes with enhanced activity and precise regulatory features. Here, we report the AlphaFold3-guided enhancement of MagMboI, a photoactivatable restriction enzyme designed for light-controlled top-down genome engineering. MagMboI is derived from the type II restriction enzyme MboI and functions through a split-protein strategy in which its N- and C-terminal fragments are fused to light-inducible dimerization modules. Upon exposure to blue light, these domains heterodimerize, restoring nuclease activity in a controlled manner. Using AlphaFold3, we modeled the structure of the MagMboI-DNA complex and gained structural insights into the interaction between MagMboI and its target DNA recognition sequence (5′-GATC-3′) required for Mg2+-dependent DNA cleavage. Comparing neighboring split-site variants, we identified an alternative split that increases the MagMboI–DNA interface area and enhances complex stability relative to the original construct. This redesigned variant (designated MagMboI-plus) preserves α-helical integrity while strengthening protein-DNA contacts. Although MagMboI-plus, when introduced in Saccharomyces cerevisiae cells, exhibited slightly increased DNA-cleavage activity in vivo upon blue light activation, it was found to induce more pronounced genomic rearrangements compared to the original MagMboI construct. These findings demonstrate that AlphaFold3-based prediction can accelerate functional improvements in engineered enzymes, providing a strategy for developing light-controlled genome engineering tools.