Elucidation of the catalytic mechanism of a single-metal dependent homing endonuclease using QM and QM/MM approaches: the case study of I-PpoI

Homing endonucleases (HEs) are highly specific DNA cleaving enzymes, with I-PpoI having been suggested to use a single metal to accelerate phosphodiester bond cleavage. Although an I-PpoI mechanism has been proposed based on experimental structural data, no consensus has been reached regarding the roles of the metal or key active site amino acids. This study uses QM cluster and QM/MM calculations to provide atomic-level details of the I-PpoI catalytic mechanism. Minimal QM cluster and large-scale QM/MM models demonstrate that the experimentally-proposed pathway involving direct Mg2+ coordination to the substrate coupled with leaving group protonation through a metal-activated water is not feasible due to an inconducive I-PpoI active site alignment. Despite QM cluster models of varying size uncovering a pathway involving leaving group protonation by a metal-activated water, indirect (water-mediated) metal coordination to the substrate is required to afford this pathway, which renders this mechanism energetically infeasible. Instead, QM cluster models reveal that the preferred pathway involves direct Mg2+–O3′ coordination to stabilize the charged substrate and assist leaving group departure, while H98 activates the water nucleophile. These calculations also underscore that both catalytic residues that directly interact with the substrate and secondary amino acids that position or stabilize these residues are required for efficient catalysis. QM/MM calculations on the solvated enzyme–DNA complex verify the preferred mechanism, which is fully consistent with experimental kinetic, structural, and mutational data. The fundamental understanding of the I-PpoI mechanism of action, gained from the present work can be used to further explore potential uses of this enzyme in biotechnology and medicine, and direct future computational investigations of other members of the understudied HE family.