Investigations into the modes of substrate binding to aminoglycoside-modifying enzymes

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 revealed two possible 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 have been found to be Φ_AB = -7.9° ± 2.0° and Ψ_AB = -46.2° ± 0.6° for conformer 1 and Φ_AB = -69.4° ± 2.0° and Ψ_AB = -57.7° ± 0.5° for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300° Kelvin. 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. The enzyme-bound conformation of butirosin A yielded a bisecting arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the stacking arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3-O-phosphotransferase [Cox, J. R., & Serpersu, E. H. (1997) Biochemistry 36,2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pK_a values for its amino groups are also reported. In addition, the cloning and overexpression of aminoglycoside 2"-(O-nucleotidyltransferase-Ia from P. aeruginosa R-plasmid is also reported.