Cloning, purification and substrate conformations of aminoglycoside acetyltransferase (3)-IIIb

Author

Michael Andrew Owston

Date of Award

8-2001

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin Serpersu

Abstract

The aminoglycoside resistance protein aminoglycoside acetyltransferase (3)-IIIb (AAC3- nib) was successfully cloned, expressed and purified. This will allow many future experiments to be done using AAC3-IIIb. Using 2D NMR and molecular modeling, the conformations of two aminoglycosides, kanamycin and ribostamycin, were determined when bound to AAC3-lIIb. The structures determined for ribostamycin were divided into two conformers defined by the phi-psi angles of the glycosidic bonds as follows: Conformer 1 (11 total): Φ_1A = -22 +/- 3, &Psi,_1A= -42 +/-1, Φ_1C= -9 +/- 4, Ψ_1C = 51 +/-1 and for Conformer 2 (11 total): Φ_1A = -67 +/- 0.7, Ψ_1A = -59 +/- 0.8, Φ_1C = -9 +/-3, Ψ_1C = 49 +/-1. This gives the C to B ring orientation with respect to one another to be the same in both conformers. For kanamycin there were three conformers determined using the phi-psi angles as follows: Conformer 1 (9 structures): Φ_1A = -11 +/- 3, Ψ_1A= - 45 +/- 3, Φ_1C= -9 +/- 4, Ψ_1C = 50 +/- 2; Conformer 2 (6 structures): Φ_1A = -8 +/- 3, Ψ_1A = 48 +/- 2, Φ_1C= -18 +/- 2, Ψ_1C = -42 +/-2; and Conformer 3 (5 structures): Phi;_1A = -73 +/- 0.6, Psi;_{1A= -57 +/- 0.2, Φ1C -14 +/- 0.5, Ψ1C = 51 +/- 0.2. Because the B to C orientation was the same for Conformer 1 and Conformer 3, this gives 14 out of 20 structures with that conformation for these rings. The C ring appears to be more constrained when either aminoglycoside is used while the A ring is somewhat more flexible. However, a comparison of the two aminoglycosides gives the A ring in one orientation (Conformer 1 for both ribostamycin and kanamycin) more than the others. This suggests that this may be the proper orientation of this ring. It has been suggested that the A and B rings provide the most important contributions when binding to both other aminoglycoside modifying enzymes and to RNA (Fourmy et al, 1996; Yoshizawa et al, 1998; Serpersu et al, 2000). Comparisons superimposing these rings with other enzyme bound aminoglycosides as well as RNA bound aminoglycosides shows remarkable similarity among the conformations. This gives important conformational information that may be used in future drug and enzyme inhibitor design studies.}

Recommended Citation

Owston, Michael Andrew, "Cloning, purification and substrate conformations of aminoglycoside acetyltransferase (3)-IIIb. " Master's Thesis, University of Tennessee, 2001.
https://trace.tennessee.edu/utk_gradthes/9703