Backbone resonance assignments of a promiscuous aminoglycoside antibiotic resistance enzyme; the aminoglycoside phosphotransferase(3′)-IIIa

Abstract

The aminoglycoside phosphotransferase(3')-IIIa (APH) is a promiscuous enzyme and renders a large number of structurally diverse aminoglycoside antibiotics useless against infectious bacteria. A remarkable property of this approximately 31 kDa enzyme is in its unusual dynamic behavior in solution; the apo-form of the enzyme exchanges all of its backbone amide protons within 15 h of exposure to D ( 2 ) O while aminoglycoside-bound forms retain approximately 40% of the amide protons even after >90 h of exposure. Moreover, the number of observable peaks and their dispersion in HSQC spectra varies with each aminoglycoside, rendering the resonance assignments very challenging. Therefore, the binary APH-tobramycin complex, which shows the largest number of well-resolved peaks, was used for the backbone resonance assignments (Calpha, C, N, H, and some Cbeta) of this protein (BMRB-16337).

Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

“…Such behavior appears to be common with aminoglycoside modifying enzymes because we also observed similar aminoglycoside-dependent spectral changes in dynamic properties of a different enzyme, the aminoglycoside phosphotransferase(3)-IIIa. 7,24 Previous data acquired by isothermal titration calorimetry (ITC) indicated that CoASH is able to increase antibiotic binding affinity to AAC in an antibioticdependent manner that varies from 4-fold up to 15-fold, 5 providing additional evidence that there is a link between these two distinct ligand binding sites. It is likely that binding of CoASH to AAC causes conformational changes in the aminoglycoside binding site, which also affects dynamic properties of some backbone amides.…”