AlphaFold3-guided optimization of a photoactivatable endonuclease for top-down genome engineering
Year: 2025
Type: article
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 Mg 2+ -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. (209/250 words)
Source: Journal of Biological Chemistry
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,430
Funders: Support for Pioneering Research Initiated by the Next Generation, Japan Agency for Medical Research and Development, Ministry of Education, Culture, Sports, Science and Technology, Japan Society for the Promotion of Science, Japan Science and Technology Agency, Core Research for Evolutional Science and Technology
OpenAlex ID: W4414579486