Isolation and substrate binding studies of aminoglycoside nucleotidyltransferase 2" Ia

Author

Drew Robert Ekman

Date of Award

8-2000

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin H. Serpersu

Committee Members

Cynthie B. Peterson

Abstract

Aminoglycoside nucleotidyltransferase (2")-la (ANT) was cloned from P Aeruginosa and purified from overexpressing E coli BL21 (DE3) cells. The high yields obtainable from this system will permit in-depth structural studies in the future. In addition, the conformation of the aminoglycoside antibiotic isepamicin, a pseudo-tri-saccharide, bound to aminoglycoside nucleotidyltransferase 2"-la has been determined using NMR spectroscopy. Molecular modeling employing experimentally determined inter-proton distances resulted in two different enzyme-bound conformations (conformer 1 and conformer 2) of isepamicin when comparing the structures using the 2-deoxystreptamine ring (B ring) and the D-garosamine ring (C ring). Conformer 1 was by far the major conformer defined by the average glycosidic dihedral angles φ_BC -65.26 ± 1 63 and Ψ_BC-54 76 ± 4 64 (20 structures) . Conformer 1 was further subdivided into one major (conformer 1a) and two minor components (conformer 1b and 1c) based on the comparison of Φ_AB and Ψ_AB. Conformer 1a and conformer 2 have almost identical Φ_AB and Ψ_AB values. In addition, further comparisons with the A and B rings were made with the conformations of isepamicin bound to both a phosphotransferase (APH (3')-llla) and an acetyltransferase (AAC(6')-li) again resulting in striking similarities. It has been suggested that aminoglycosides use both electrostatic interactions as well as hydrogen bonds in binding to these enzymes and that the contacts made by the A and B rings to active site residues are the most crucial (Serpersu et. al , 2000). The similarities in isepamicin bound to three different aminoglycoside modifying enzymes lends further support to this theory. This information should aid in the design of effective inhibitors possessing a broad range of aminoglycoside modifying enzymes as targets.

Recommended Citation

Ekman, Drew Robert, "Isolation and substrate binding studies of aminoglycoside nucleotidyltransferase 2" Ia. " Master's Thesis, University of Tennessee, 2000.
https://trace.tennessee.edu/utk_gradthes/9307