Role of Asp190 in the Phosphorylation of the Antibiotic Kanamycin Catalyzed by the Aminoglycoside Phosphotransferase Enzyme: A Combined QM:QM and MD Study.

The aminoglycoside phosphotransferase (APH(3')-IIIa) kinases form a clinically central group of antibiotic-resistant enzymes. Computationally, we have studied the catalytic mechanism of the APH(3')-IIIa enzyme at the atomic-level. The proposed reaction mechanism involves protonation of Asp190 by the kanamycin 3'-hydroxyl group mediated through an explicit neighboring water molecule, which leads to a simultaneous nucleophilic attack on the γ-phosphate of the ATP by the deprotonated kanamycin 3'-hydroxyl group. The second step is a proton abstraction from the protonated Asp190 to the phosphate group of the phosphorylated kanamycin mediated by an explicit water molecule. The calculated Gibbs energy of activation (Δ) of the rate-determining step for the phosphorylation reaction is 77 kJ mol at the M06-2X/6-311++G(2df,p)//ONIOM(M06-2X/6-31+G(d):HF/6-31G(d)) level of theory. This study has provided a new understanding of the APH(3')-IIIa catalytic mechanism that agrees with the available experimental data (Δ = 75 ± 4 kJ mol) and could provide a starting point for the rational design of mechanism-based inhibitors of aminoglycoside modifying enzyme to circumvent antibiotic resistance.