Solution-Phase Nuclear Magnetic Resonance Studies of a Nonribosomal Peptide Synthetase Adenylation Domain, of a Bacterial Glycosyltransferase, and the Rational Design of Inhibitors and Mutants of Glycosyltransferases

Abstract

A molecule's biological function is determined by its chemical structure and its three dimensional (3D) shape. While a molecule's chemical structure is fairly static its physical 3D structure is typically very dynamic and thus more difficult to determine. A protein's 3D structure is actually an ensemble of shapes that it can assume depending on its immediate surroundings. The two main methods of determining a protein's 3D structure at high resolution are X-ray crystallography and nuclear magnetic resonance (NMR). These two methods complement each other by allowing for a protein's 3D shape to be studied in a wider variety of environments than either one alone can do. We are working to develop new methods for determining the 3D structures of proteins in solution by NMR, with and without ligands present that may bind to them. In particular we are developing NMR methods for studying the solution-phase 3D structures of large, biologically important, enzymes. We are interested in determining the solution-phase 3D structures of enzymes at the atomic level so that we can understand their biological functions and how they accomplish them, and thus how to control them in order to treat diseases and improve human health. We are also interested in using high resolution structures of enzymes to do structure-based reengineering of them. Redesigning enzymes enhances our understanding of how they function in their native environment and leads to redesignedversions of them that can be used to chemoenzymatically synthesize clinically important drugs. This dissertation begins with our studies, by NMR, of the solution-phase structures of two bacterial enzymes involved in the biosynthesis of antibiotics. In particular we studied the solution-phase structures of the adenylation domain responsible for selectively activating the amino acid phenylalanine in the biosynthetic pathway for the antibiotic gramicidin S. Next, we present our studies of two glycosylation enzymes involved in the final phase of biosynthesis of the antibiotic vancomycin. We compared two approaches to determine the amino acids involved in substrate binding by these two enzymes, a solution-phase NMR approach and an in silico protein modeling, with ligand docking, approach. These enzymes are each quite large for current NMR solution-phase techniques and we present the lessons we learned from studying them and our plans for future work. Finally, we present a review of the use of small-molecule inhibitors and enzyme redesign in the study of the function of glycosyltransferases, with applications in the treatment of glycosylation disorders in humans and the chemoenzymatic synthesis of homogeneously glycosylated molecules.