Doctoral Dissertations

Date of Award

12-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin H. Serpersu

Committee Members

Cynthia B. Peterson, Wesley D. Wicks, Solon Georghiou

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 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 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° 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 pKa 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.

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