Aminoglycoside-Antibiotic Modifying Enzymes

University Of Tennessee Knoxville, Knoxville TN

Investigators

Abstract

Enzyme-substrate interactions between the aminoglycoside antibiotics and aminoglycoside modifying enzymes are very interesting and challenging targets for basic research. Aminoglycoside antibiotics constitute a large group of clinically important drugs, and today, there are more than fifty known enzymes that chemically modify aminoglycosides and render them useless against pathogenic bacteria. These enzymes fall into three groups based on the type of chemical modification they catalyze. These reactions are N-acetylation, O-adenylation, and O-phosphorylation. Most notably, each enzyme can use many aminoglycosides as substrates; conversely a single aminoglycoside can be modified by a large number of enzymes. This situation makes the structural and functional studies quite challenging and currently neither a solution structure of an aminoglycoside-modifying enzyme, nor a crystal structure of an enzyme that yields information on the conformation of a bound aminoglycoside is available. This research will determine the first solution structures of aminoglycoside-modifying enzymes by NMR. The enzymes are the aminoglycoside-2'-nucleotidyltransferase-Ia, and the aminoglycoside-3-acetyltransferase-IIIb. NMR and other biochemical and biophysical methods will be used to study enzyme-antibiotic interactions in detail. The specific aims include kinetic and binding studies, determination of structures of both enzymes with and without substrates, substrate titrations using isotope-enriched enzymes or substrates, site specific mutations of the critical residues, measurement of amide exchange rates with and without substrates. It is expected that the solution structures of an aminoglycoside 2'-adenyltransferase, and an aminoglycoside 3-acetyltransferase will be determined. It is also expected that kinetic and thermodynamic properties of enzyme-substrate complexes, the residues involved in substrate binding and catalysis, conformations of tightly bound aminoglycosides, and aminoglycoside-enzyme contact site(s)will be studied. It is expected that these studies will reveal the types of interactions that occur between enzymes and aminoglycosides, thus allowing the determination of shared characteristic features of the active sites of these enzymes that allow binding of aminoglycosides in a similar conformation. This can be significant since the homology between some of the aminoglycoside-modifying enzymes can be very little or none. In general, this type of a system allows one to compare conformations of many ligands bound to the same target site as well as the comparison of active site structures of different targets (enzymes) that have no homology to each other but provide the same complementary groups to bind the same ligands in the same conformation. Data obtained in such comparisons should be helpful to determine the essential principles that affect ligand-macromolecule interactions. Furthermore, it should also lead to predictions on the expected binding modes of structurally related compounds and change in their affinities as a result of mutations at the active site(s). Results of this work should yield a better understanding of enzyme-ligand interactions and may have far reaching impact on studies of many ligand-receptor interactions.