The  aminoglycoside phosphotranferase (3')-IIIa (APH) is a broad spectrum enzyme that is capable of modifying more than a dozen, structurally diverse aminoglycoside antibiotics.  NMR spectrum of the apo enzyme shows a highly dynamic enzyme in solution. The thermodynamics of unfolding of APH have been studied by differential scanning calorimetry. The apo protein reversibly unfolds at 46�C with an unusually low heat capacity change. The melting temperature is increased by antibiotic substrates in a concentration-dependent manner with an enthalpy change reflecting both protein denaturation and ligand release (i.e., no additional �stabilization� of the enzyme by the ligand binding). Circular dichroism spectra show that unfolded APH retains some secondary structure elements and isothermal titration calorimetry experiments show that the unfolded enzyme can still bind aminoglycosides with a high affinity. NMR data showed that completely denatured enzyme refolds under the same conditions.

Energetics of protein stability: A highly promiscuous, antibiotic resistance causing enzyme

248th National Meeting (2014)

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