Abu-Saleh Abd Al-Aziz A, Sharma Sweta, Yadav Arpita, Poirier Raymond A
Chemistry Department, Memorial University, St. John's, Newfoundland and Labrador A1B 3X7, Canada.
Department of Chemistry, University Institute of Engineering & Technology, Chhatrapati Shahu Ji Maharaj University, Kanpur 208024, India.
J Phys Chem B. 2020 Apr 30;124(17):3494-3504. doi: 10.1021/acs.jpcb.0c01604. Epub 2020 Apr 17.
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.
氨基糖苷磷酸转移酶（APH(3')-IIIa）激酶构成了临床上一类核心的抗生素抗性酶。通过计算，我们在原子水平上研究了APH(3')-IIIa酶的催化机制。提出的反应机制涉及卡那霉素3'-羟基通过一个明确的相邻水分子介导使Asp190质子化，这导致去质子化的卡那霉素3'-羟基同时对ATP的γ-磷酸进行亲核攻击。第二步是由一个明确的水分子介导，将质子化的Asp190上的质子夺取到磷酸化卡那霉素的磷酸基团上。在M06-2X/6-311++G(2df,p)//ONIOM(M06-2X/6-31+G(d):HF/6-31G(d))理论水平下，磷酸化反应速率决定步骤的计算活化吉布斯自由能（Δ）为77 kJ mol 。这项研究为APH(3')-IIIa催化机制提供了新的认识，与现有实验数据（Δ = 75 ± 4 kJ mol）相符，并可为基于机制的氨基糖苷修饰酶抑制剂的合理设计提供起点，以规避抗生素耐药性。
