Solution studies of isepamicin and conformational comparisons between isepamicin and butirosin A when bound to an aminoglycoside 6'-N-acetyltransferase determined by NMR spectroscopy.
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-acetyltransferase-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 phi AB = -7.9 +/- 2.0 degrees and psi AB = -46.2 +/- 0.6 degrees for conformer 1 and phi AB = -69.4 +/- 2.0 degrees and psi AB = -57.7 +/- 0.5 degrees 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) Biochemistry 36, 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.
DiGiammarino
E L
EL
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville 37996-0840, USA.
Draker
K A
KA
Wright
G D
GD
Serpersu
E H
EH
D000123:Acetyltransferases / Q000378:metabolism*; D001665:Binding Sites; D002076:Butirosin Sulfate / Q000737:chemistry* / Q000378:metabolism; D016984:Enterococcus faecium / Q000201:enzymology; D005839:Gentamicins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D012996:Solutions; D013379:Substrate Specificity
D005839:Gentamicins; D012996:Solutions; D002076:Butirosin Sulfate; D000123:Acetyltransferases; C056532:aminoglycoside N(6')-acetyltransferase; C016927:isepamicin