UNIVERSITY OF TENNESSEE
Grant: $782,240 - National Science Foundation - Jul. 7, 2009
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Award Description: This project is designed to dissect dynamic, thermodynamic, and structural properties of various enzyme–aminoglycoside complexes with the ultimate goal of understanding the molecular properties of these promiscuous enzymes as a whole group. Aminoglycosides are a large group of antibiotics produced by actinomycetes as a defensive measure against other bacteria. To avoid effects of these antibiotics, they also produce enzymes that modify aminoglycosides (AGMEs) and render them harmless. A large majority of these enzymes (more than fifty known today) are capable of modifying a number of aminoglycosides; conversely a single aminoglycoside can be modified by a large number of enzymes. Such intertwined interactions between these molecules render the structural and functional studies quite challenging and detailed studies directed toward the understanding of molecular properties common to all or most aminoglycoside-modifying enzymes can only be studied by selecting a representative set to study due to the large size of this family of enzymes. Therefore, this project is designed to study a representative enzyme from each of the three groups that catalyze a different reactions with aminoglycoside antibiotics and study kinetic, thermodynamic, dynamic, and structural properties of enzyme–ligand complexes in molecular detail with these enzymes. The three enzymes with different substrate profiles include an acetyltransferase, the aminoglycoside acetyltransferase(3)-IIIb (AAC), a nucleotidyltransferase, the aminoglycoside nucleotidyltransferase(2')-Ia (ANT), and a phosphotransferase, the aminoglycoside phosphotransferase(3?)-IIIa (APH). AAC catalyzes transfer of the acetyl group from Acetyl Coenzyme A to the 3-NH2 of aminoglycoside antibiotics. ANT catalyzes nucleotidyl transfer to the 2'-OH of aminoglycosides from MgATP. APH phosphorylates the 3'-OH (and also 5'-OH in neomycins) of aminoglycoside antibiotics. These enzymes modify important sites on aminoglycosides, which are remote from each other. They have no sequence homology to each other, and have significantly different substrate profiles and yet, there are several aminoglycoside antibiotics that are substrates for all three enzymes. APH does not discriminate between kanamycins and neomycins and has the broadest substrate specificity among the phosphotransferases. AAC can modify neomycins and kanamycins but shows much higher catalytic activity with kanamycins and has a narrower substrate range compared to APH. ANT has the most selective substrate profile of the three such that it does not modify neomycins. These enzymes, therefore, represent an excellent model system to study the general molecular principles of aminoglycoside-enzyme interactions and molecular basis of their substrate selectivity. This project also involves efforts to bridge computational and experimental aspects of a biologically significant and challenging problem. Identification of sites on ligands and proteins that are affected by the global properties of the enzyme versus those affected only by sub-global and local structural perturbations may yield valuable clues to understand promiscuity of these enzymes. Computational studies to determine dynamic properties of the enzymes and ionization state of functional groups will be performed. Binding of aminoglycosides to enzymes will be studied by variety of biophysical techniques including fluorescence spectroscopy, isotthermal titration calorimetry, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Combined use of experimental and computational data will lead to design new strategies to expand horizon of both approaches as applied to enzyme–ligand complexes in general.
Project Description: This project is started in August 1, 2009. One of the specific aims of this project is the characterization of kinetic and thermodynamic properties of enzyme-antibiotic complexes of the antibiotic resistance enzyme the aminoglycoside acetyltransferase(3)-IIIa. Kinetic studies showed that the enzyme catalyzes the reactions of kanamycin group aminoglycosides faster than those with neomycin group aminoglycosides. Currently, we are performing isothermal titration calorimetry (ITC) experiments to determine thermodynamic parameters of complexes of this promiscuous enzyme with a number of aminoglycoside antibiotics that it acetylates and cause resistance to their action in bacteria. Initial studies with several aminoglycoside antibiotics revealed that binding of antibiotics to this enzyme occurs with favorable enthalpy and unfavorable entropy yielding an overall favorable free energy of binding. The presence of co-substrate analog coenzyme A (CoA) increases the affinity of aminoglycosides to this enzyme by making the enthalpy of binding more favorable. Conversely, the presence of the aminoglycoside also increases the affinity of CoA to the enzyme. Since the binding of aminoglycosides to the enzyme causes shifts in protonation/deprotonation equilibrium of various functional groups on the enzyme and antibiotics, these studies must be performed in different buffer solutions with different heat of ionization to determine the extent of protonation and the true intrinsic enthalpy of binding. These studies are now in progress. Additional set of studies are being performed to characterize the reaction product and confirm that the acetylation of aminoglycosides occur at the N3 position. These studies are performed by nuclear magnetic resonance spectroscopy.
Infrastructure Description: This is a conditional field. This grant is not an Infrastructure project.
Jobs Summary: This project will support graduate students and postdoctoral scientists. We are currently interviewing candidates postdoctoral scientist position. A graduate student in the PI’s laboratory will also be supported by this award. Currently she is supported by a different fellowship. Her support will start in the spring semester. (Total jobs reported: 0)
Project Status: Less Than 50% Completed
This award's data was last updated on Jul. 7, 2009. Help expand these official descriptions using the wiki below.
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