Enzymes containing pyridoxal-5'-phosphate (PLP) are involved in a broad range of reactions of amino acids and amines, including transamination, racemization, decarboxylation, ?- and ?-elimination, ?- and ?- substitution, and, as recently discovered, even oxidation and oxygenation. A number of important current or prospective drug targets are PLP-dependent enzymes, including ?-aminobutyrate aminotransferase, DOPA decarboxylase, alanine racemase, ornithine decarboxylase, and serine hydroxymethyltransferase. However, many of the current drugs that target PLP-dependent enzymes suffer from side effects due to lack of specificity for their targets. Thus, it is important to understand the reactions of these enzymes with molecular and atomic levels of detail to help in the design of new more potent and more selective drugs. Using X-ray crystallography, a great deal has been learned about the role of both enzymes and cofactor in catalysis. Despite this, there are still critical gaps in our understanding of PLP-dependent enzymes that limit drug design. Crystal structures alone are missing two essential pieces of information. First, they lack important information regarding reaction dynamics. Protein motion in ligand binding and catalysis is known to play a central role in enzymes, but how this occurs is essentially unknown. In addition, hydrogen atoms that play critical roles in PLP catalysis are not directly observed by X-ray crystallography. This leaves a significant gap in our understanding of general acid-base catalysis in enzymes in general and particularly in PLP-dependent enzymes, where active site protonation states appear to play critical roles in control of reaction specificity. A recent neutron diffraction structure of aspartate aminotransferase found a proton in an unpredicted position in the active site, forming a low barrier hydrogen bond between the substrate carboxylate and the aldimine nitrogen. This void in our understanding of protonation and ionization states impedes rational design of therapeutic agents that, for example, are tailored for specific electrostatic environments. The goal of the proposed project is to provide a very detailed understanding of PLP enzyme mechanisms by coordinately defining their structures and dynamics from the global to the atomic level. To accomplish this, we will employ a synergistic combination of biophysical techniques that are sensitive to different size- and time-scales. These will include joint X-ray/neutron crystallography, solid-state NMR crystallography, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations, inelastic neutron scattering, steady-state and rapid kinetics techniques of PLP dependent enzymes. The results of this collaborative venture will provide, for the very first time, a global picture of catalysis by a large and centrally important class of enzymes at true atomic-resolution for stable intermediates as well as the dynamic connections between them. The insights from our results and the techniques developed will be transferable to many other enzymes, and may contribute to improved rational drug design of novel antibiotic, antidiabetic, antimalarial, and other drugs.
Structural and proton dynamics of pyridoxal-5?-phosphate dependent enzymes The goal of the proposed project is to provide a very detailed understanding of pyridoxal-5?-phosphate (vitamin B6) dependent enzyme mechanisms by coordinately defining their structures and dynamics from the global to the atomic level. To accomplish this, we will employ a synergistic combination of biophysical techniques that are sensitive to different size-scales, from protons to proteins, and time- scales, from picoseconds to minutes. The insights from our results and the techniques developed will be transferable to many other enzymes, and may contribute to improved rational drug design of novel antibiotics, antidiabetics, antimalarials, and other drugs.
