Structural and Functional Characterization of a Novel Aminoglycoside-Modifying Enzyme from NDM-1-Producing Enterobacteriaceae in Nigerian Hospital Environments

Aminoglycoside-modifying enzymes (AMEs) are a critical family of resistance determinants that chemically inactivate aminoglycoside antibiotics through acetylation, phosphorylation, or adenylation. The global spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Enterobacteriaceae frequently co-selects for AME genes on mobile plasmids, combining carbapenem and aminoglycoside resistance in single organisms. A novel AME gene variant was detected in silico during resistome annotation of NDM-1-producing Klebsiella pneumoniae from hospital effluent samples in Abuja, warranting full structural and functional characterization. This dissertation presents the structural and functional characterization of a novel AME variant (provisionally designated Aac(3)-VId) from NDM-1-producing Enterobacteriaceae isolated from hospital environments across the Federal Capital Territory and Niger State. The Aac(3)-VId gene was cloned into pET-28a expression vector and heterologously expressed in E. coli BL21(DE3). The recombinant protein was purified by Ni-NTA affinity chromatography. Enzymatic activity was characterized by spectrophotometric kinetics assays (Km, Vmax, kcat) against a panel of 12 aminoglycosides. Crystal structure was determined at 1.85 angstrom resolution by X-ray crystallography using synchrotron beamline data. Molecular docking of aminoglycoside substrates was performed using Glide SP docking in Schrodinger Suite. Aac(3)-VId demonstrated acetyltransferase activity against gentamicin, tobramycin, and amikacin, with amikacin representing a newly identified substrate not documented in the reference Aac(3) family. The crystal structure revealed a distinctive substrate-binding loop conformation absent in all homologs, explaining the expanded substrate range. Molecular epidemiology data identified Aac(3)-VId on an IncFII plasmid co-carrying blaNDM-1, representing a clinically significant resistance gene combination. Keywords: aminoglycoside-modifying enzyme, AME, NDM-1, crystal structure, Abuja.