A co-evolving active site network regulates cleavage preference in LAGLIDADG homing endonucleases

Evolutionary pathways leading to innovation in enzyme substrate recognition and catalysis are poorly understood. Here, we use LAGLIDADG homing endonucleases (meganucleases) to examine the relationship between variation in active site residues and catalysis that is regulated by indirect readout of the central 4 bases of DNA substrates. Functional selections and deep sequencing profiled the fitness landscape of 1600 variants of a co-evolving active site network individually challenged with 67 substrates poorly cleaved by the meganucleases I-LtrI and I-OnuI. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild type proteins co-crystallized with substrate containing the non-cognate AACC central 4 bases revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Our data indicate that the E184D substitution broadens cleavage specificity by accommodating indirect readout of a diverse range of DNA substrates as compared to the wild-type enzyme. This flexibility allows meganucleases to fine-tune DNA-substrate interactions on an evolutionary timescale to promote persistence as mobile elements. Our study provides a workflow to modulate the cleavage range of re- purposed meganucleases or other site-specific endonucleases.