Theoretical Studies on Mechanism of Inactivation of Kanamycin A by 4'-O-Nucleotidyltransferase.

Sergio Martí,Katarzyna Świderek,Agatha Bastida

doi:10.3389/fchem.2018.00660

Abstract

This work is focused on mechanistic studies of the transfer of an adenylyl group (Adenoside-5′-monophosfate) from adenosine 5′-triphosphate (ATP) to a OH-4′ hydroxyl group of an antibiotic. Using hybrid quantum mechanics/molecular mechanics (QM/MM) techniques, we study the substrate and base-assisted mechanisms of the inactivation process of kanamycin A (KAN) catalyzed by 4′-O-Nucleotidyltransferase [ANT(4′)], an active enzyme against almost all aminoglycoside antibiotics. Free energy surfaces, obtained with Free Energy Perturbation methods at the M06-2X/MM level of theory, show that the most favorable reaction path presents a barrier of 12.2 kcal·mol−1 that corresponds to the concerted activation of O4′ from KAN by Glu145. In addition, the primary and secondary 18O kinetic isotope effects (KIEs) have been computed for bridge O3α, and non-bridge O1α, O2α, and O5′ atoms of ATP. The observed normal 1°-KIE of 1.2% and 2°-KIE of 0.07% for the Glu145-assisted mechanism are in very good agreement with experimentally measured data. Additionally, based on the obtained results, the role of electrostatic and compression effects in enzymatic catalysis is discussed.

Highlights

Aminoglycoside antibiotics (AGAs) belong to the class of agents used to treat serious infections caused by bacteria that either multiply very quickly or are difficult to treat
Based on analysis made for this distance it is observed that the change of AMPCPP to adenosine 5′-triphosphate (ATP) reduces the presence of non-reactive conformations to 15% of the entire population of structures generated along the molecular dynamic (MD)
In this work the molecular mechanism of the transfer of the adenylyl group from ATP to kanamycin A (KAN), and formation of KAN-AMPMg ternary complex as a result of the reaction catalyzed by the aminoglycoside nucleotidyltransferases (ANTs)(4′) enzyme was investigated

Summary

Introduction

Aminoglycoside antibiotics (AGAs) belong to the class of agents used to treat serious infections caused by bacteria that either multiply very quickly or are difficult to treat. Their role is to stop bacteria from producing proteins needed for their survival (Shaw et al, 1993). Due to widespread usage of these antibiotics in clinical treatment, bacterial strains have appeared that make these compounds ineffective. The major mechanism of bacterial resistance in clinical isolates of gram-negative and gram-positive bacteria is assigned to enzymatic modification of the amino or hydroxyl groups of AGAs (Vakulenko and Mobashery, 2003)

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Conclusion