Unraveling the Crucial Role of Single Active Water Molecule in the Oxidative Cleavage of Aliphatic C-C Bond of 2,4 '-Dihydroxyacetophenone Catalyzed by 2,4 '-Dihydroxyacetophenone Dioxygenase Enzyme: A Quantum Mechanics/Molecular Mechanics Investigation

2,4'-Dihydroxyacetophenone dioxygenase (DAD), a nonheme dioxygenase enzyme, shows exquisite selectivity in the aliphatic C-C bond cleavage of 2,4'-dihydroxyacetophenone (DHAP) in the presence of molecular oxygen (O-2). Molecular dynamics simulations revealed the presence of a single water molecule at the active site of the enzyme. This lone water molecule is pivotal for facilitating the oxidative cleavage of the aliphatic C-C bond of 2,4'-DHAP catalyzed by DAD enzyme, as evident from the findings of our hybrid quantum mechanics/molecular mechanics (QM/MM) studies. 2,4'-DHAP is initially deprotonated through a relay proton transfer mechanism with the aid of the active site water molecule. This water molecule also actively participates in the O-O and C-C bond cleavage steps. The activated water molecule acts as catalytic acid base species. The O-O cleavage step has been predicted to be the rate-determining step with an associated barrier of 20.3 kcal/mol calculated at the uB3LYP-D3/def2-TZVP/OPLS level of theory on the quintet spin surface. Multiple sequence alignment of the bacterial DAD enzyme has shown the evolutionary importance of the Tyr93 and Glu108 residues, in which Tyr93 acts as proton carrier and G1u108 acts as reservoir, during the relay proton transfer. Our study demonstrates the active role of water in the catalytic cycle of DAD enzyme and additionally unearthed important indirect roles of the two amino acids (Tyr93 and Glu108) in the enzymatic cycle.