Isolation and Thermodynamic Characterization of Aminoglycoside Nucleotidyltransferase (2″)-Ia

Author

Roger Edward Wright, University of Tennessee - Knoxville

Date of Award

12-2005

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin Serpersu

Committee Members

David Baker, Elias Fernandez, Cynthia Peterson

Abstract

Aminoglycoside nucleotidyltransferase(2″)-Ia is one of the most important aminoglycoside-modifying enzymes. Because of the difficulties in isolating this enzyme with a high level of purity, very little work has been reported for this enzyme. A procedure for obtaining this enzyme with a purity level of greater than 95% from inclusion bodies was developed. The optimal conditions for isolating ANT(2″) were solubilizing the inclusion bodies in 8 M urea followed by direct dilution of the enzyme into 0.1 M Tris-HCl pH 8.5, 0.2 M KCl, 0.4 ML-arginine and 5 mM reduced glutathione at 4°C. The determination of an effective method of obtaining this enzyme allowed for characterization by thermodynamic and kinetic methods. Fluorescence spectroscopy titrations showed that ANT(2″) binds MgATP much tighter than free ATP and electron paramagnetic resonance (EPR) experiments showed that the enzyme binds a second divalent cation in addition to the one in the MgATP complex. Isothermal titration calorimetry (ITC) was used to show that binding of metal –nucleotide increases the affinity for the aminoglycoside substrate for all but one aminoglycoside. The binding of aminoglycosides to ANT(2″) occurs with a favorable negative enthalpy and unfavorable

negative entropy. Molecular determinants of substrate specificity for aminoglycosides were also determined. An amino group at the 2 ′ position is preferred over a 2′-hydroxyl in terms of greater catalytic efficiency (kcat/Km) and tighter binding. The same preference is observed at the 6 ′ position where an amino group rather than a hydroxyl at that position leads to tighter binding. The 1 position on the deoxystreptamine ring is also shown to be important in terms of binding and catalysis.

Recommended Citation

Wright, Roger Edward, "Isolation and Thermodynamic Characterization of Aminoglycoside Nucleotidyltransferase (2″)-Ia. " PhD diss., University of Tennessee, 2005.
https://trace.tennessee.edu/utk_graddiss/2314