Structural and proton dynamics of pyridoxal-5’-phosphate dependent enzymes Resubmission (Diversity Supplement)

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Project Summary (NO CHANGE IN THE SCOPE OF THE PARENT PROJECT) PLP-dependent enzymes represent about 2% of the proteome, and a number of them are current or potential drug targets. There are four major families of pyridoxal-5’-phosphate (PLP)-dependent enzymes, distinguished by different three-dimensional folds: the aspartate aminotransferase or α-family (Fold I), the tryptophan synthase or β-family (Fold II), the alanine racemase family (Fold III), and the D-amino acid aminotransferase family (Fold IV). Using X-ray crystallography, a great deal has been learned about the role of both these enzymes and cofactor in catalysis. Despite this, there are still critical gaps in our understanding that limit drug design. The goal of the proposed project is to provide a very detailed understanding of PLP-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- and time-scales. These will include joint X-ray/neutron crystallography, solid-state NMR spectroscopy, molecular dynamics and QM/MM calculations, inelastic neutron scattering, rapid kinetics techniques, and heavy enzyme kinetic isotope effects. We will focus on four structurally well-characterized PLP-dependent enzymes, aspartate aminotransferase, serine hydroxymethyltransferase, tyrosine phenol-lyase and tryptophan synthase, but for which information on protonation and dynamics is lacking. The enzymes are drug targets (aspartate aminotransferase, serine hydroxymethyltransferase) or serve as models for drug targets (tryptophan synthase, tyrosine phenol-lyase). These enzymes catalyze diverse reactions, but use the same cofactor in similar active sites. Thus, we postulate that the reaction specificity must be controlled by a combination of protein dynamics and selective protonation of reaction intermediates. Joint X-ray/neutron crystallography will be the foundation of our research, providing an atomic-level structural basis for protein dynamics and accurate visualization of hydrogen atoms in protein structures at moderate resolutions. The results of X-ray/neutron crystallography will be combined with novel solid-state NMR crystallography and with inelastic neutron scattering to characterize the global and local motions of these enzymes at picosecond-to- microsecond time scales. Pressure-jump relaxation kinetics, and heavy enzyme kinetic isotope effects will complement and provide dynamic information on domain motion in the picosecond to minute time-scales. It should be noted that the inelastic neutron scattering, pressure-jump and heavy enzyme kinetics are complementary techniques in that they all are sensitive to changes in the vibrational motions of the enzyme, but interrogate at different time scales. All these results will be integrated with molecular computations to provide an unprecedented complete picture of the dynamic properties of these important enzymes. This knowledge may allow the design of novel, potent and selective enzyme inhibitors that may provide new drugs targeted against PLP-dependent enzymes.