Journal of Biological Chemistry

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.

AlphaFold3

artificial intelligence

protein structure

protein engineering

optogenetics

endonuclease

restriction enzyme

DNA damage

DNA repair

genome rearrangement

break-induced replication

Burrows-Wheeler Aligner

C-terminal fragment of MboI

flavin adenine dinucleotide

FK506-binding protein

FKBP-rapamycin-binding domain of mTOR

green fluorescent protein

Integrative Genomics Viewer

loss of heterozygosity

light-oxygen-voltage domain

AlphaFold3-improved MagMboI variant

non-allelic homologous recombination

non-homologous end joining

nuclear localization signal

NHEJ-mediated translocation

N-terminal fragment of MboI

negative Magnet domain

predicted local distance difference test

positive Magnet domain

polymerase chain reaction

quantum mechanics/molecular mechanics

solvent-accessible surface area

synthetic defined/monosodium glutamate

single-nucleotide polymorphism

single-nucleotide variation

yeast extract-peptone-dextrose

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé