The use of neamine as a molecular template: Identification of active site residues in the bacterial antibiotic resistance enzyme aminoglycoside 3′-phosphotransferase type IIa by mass spectroscopy

Abstract

Four novel aminoglycoside-based affinity inactivators were shown to covalently modify the active site of aminoglycoside 3′-phosphotransferase type IIa (APH(3′)-IIa), an important resistance factor in bacteria for aminoglycoside antibiotics. Standard peptide mapping techniques failed with this enzyme. A novel mass spectroscopic analysis which combines protease digestion on the instrument probe, followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described which permitted rapid identification of the sites of protein modification. By this new technique, Glu-3 and Asp-23 were identified as active-site residues, the side chains of which potentially may serve as counter ions for the ammonium functionalities at positions 6′, and 1 and 3 of the antibiotic substrates, respectively. These findings contradict previous assertions that the C-terminal third of the enzyme should form the active site, by placing the active site clearly in the N-terminal portion of the enzyme.