Solution Studies of Isepamicin and Conformational Comparisons between Isepamicin and Butirosin A When Bound to an Aminoglycoside 6‘-N-Acetyltransferase Determined by NMR Spectroscopy

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Abstract
NMR spectroscopy, combined with molecular modeling, was used to determine the conformations of isepamicin and butirosin A in the active site of aminoglycoside 6‘-N-acetyltranferase-Ii [AAC(6‘)-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6‘)-Ii-bound isepamicin were φAB = −7.9 ± 2.0° and ψAB = −46.2 ± 0.6° for conformer 1 and φAB = −69.4 ± 2.0° and ψAB = −57.7 ± 0.5° for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6‘ nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6‘)-Ii-bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-d-glucose and d-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3‘-O-phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry36, 2353−2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pKa values for it's amino groups are also reported.