Expanding Aminoglycoside Resistance Enzyme Regiospecificity by Mutation and Truncation.

Abstract

Aminoglycosides (AGs) are broad-spectrum antibiotics famous for their antibacterial activity against Gram-positive and Gram-negative bacteria, as well as mycobacteria. In the United States, the most prescribed AGs, including amikacin (AMK), gentamicin (GEN), and tobramycin (TOB), are vital components of the treatment for resistant bacterial infections. Arbekacin (ABK), a semisynthetic AG, is widely used for the treatment of resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in Asia. However, the rapid emergence and development of bacterial resistance are limiting the clinical application of AG antibiotics. Of all bacterial resistance mechanisms against AGs, the acquisition of AG-modifying enzymes (AMEs) by bacteria is the most common. It was previously reported that a variant of a bifunctional AME, the 6'-N-AG acetyltransferase-Ie/2″-O-AG phosphotransferase-Ia [AAC(6')-Ie/APH(2″)-Ia], containing a D80G point mutation and a truncation after amino acid 240 modified ABK and AMK at a new position, the 4‴-amine, therefore displaying a change in regiospecificity. In this study, we aimed to verify the altered regiospecificity of this bifunctional enzyme by mutation and truncation for the potential of derivatizing AGs with chemoenzymatic reactions. With the three variant enzymes in this study that contained either mutation only (D80G), truncation only (1-240), or mutation and truncation (D80G-1-240), we characterized their activity by profiling their substrate promiscuity, determined their kinetics parameters, and performed mass spectrometry to determine how and where ABK and AMK were acetylated by these enzymes. We found that the three mutant enzymes possessed distinct acetylation regiospecificity compared to that of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme and the functional AAC(6')-Ie domain [AAC(6')/APH(2″)-1-194].