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].

Plasticity of Aminoglycoside Binding to Antibiotic Kinase APH(2″)-Ia

“…With sufficient selective pressure, the enzyme may adapt and elaborate upon this activity, resulting in new, potentially clinically important antibiotic resistance. This echoes the study of Holbrook and Garneau-Tsodikova on the expansion of the other domain of this bifunctional enzyme through mutation and truncation that leads to increased acetylation of arbekacin (31). As in their work, we observed that the mutated protein does not undergo changes that lead to a substantive difference in mechanism but simply exhibits weak-affinity alternative binding modes.…”

Amikacin: Uses, Resistance, and Prospects for Inhibition

“…A small number of 6′- N -acetyltransferases with the AAC(6′)-I profile were found in Gram-positive bacteria [ 184 , 188 , 189 ]. The AAC(6′)-Ie enzyme is fused to the N -terminal end of the phosphotransferase APH(2″)-Ia, forming a bifunctional enzyme coded for by the aac(6′)-Ie-aph(2 ″ )-Ia fusion gene usually located within Tn 4001 -like transposons in Gram-positive bacteria [ 182 , 183 , 190 , 191 , 192 , 193 , 194 ]. These transposons have been found in plasmids as well as chromosomes of Gram-positive pathogens such as S. aureus , S. epidermidis , or Enterococcus faecalis [ 195 , 196 , 197 , 198 ].…”

Development of 6′-N-Acylated Isepamicin Analogs with Improved Antibacterial Activity against Isepamicin-Resistant Pathogens

“…Other enzymatic approaches to generate N -acylated AGs using AMEs have been reported. Two AG acetyltransferases (AACs), including 3- N -AG acetyltransferase AAC(3) and AAC(6′)-APH(2″) with 6′- N -acetyltransferase and 2″- O -phosphotransferase activities, exhibited promiscuity towards a number of AGs, as well as diverse acyl-CoAs, resulting in the generation of novel mono- and hetero-di- N -acylated AGs [ 11 , 12 , 13 ]. However, the biological activities of most resulting analogs against resistant bacterial pathogens have not been reported.…”