The goal of this proposal is to provide the chemical level details necessary to understand the enzymatic mechanism in tryptophan synthase at atomic resolution. Enzymes have evolved to achieve remarkably efficient and specific chemical transformations that enable a diverse biochemistry. Yet atomic level details of enzyme mechanisms remain elusive; the intermediates are transient and the chemistry that drives the transformation, such as changes in hyrbridizaton and protonation states, is difficult to characterize in functioning enzyme systems. The pyridoxal-phosphate (vitamin-B6)-dependent tryptophan synthase 1222 bienzyme complex catalyses the last two steps in the synthesis of L-Trp, consecutive processes that require channeling of the common metabolite, indole, between the 1- and 2-subunits. Tryptophan synthase homologues are found in bacteria, yeasts, molds, plants, and some protozoans. The absence of a synthetic pathway for L-Trp in higher animals and in humans makes the tryptophan synthase nanomachine a potential target both for the development of herbicides, and for the design of drugs to treat infectious disease. Consequently, understanding the catalytic mechanism could provide useful insights for developing tryptophan synthase as an important target for drug design, or for the development of herbicides. Recent X-ray structure determinations of complexes with substrates, intermediates, and substrate analogues have resulted in a significant breakthrough concerning identification of the linkages between the bienzyme complex structure and catalysis. This effort combined organic synthetic work and solution kinetic/spectroscopic studies with X-ray crystal structure determinations of 10-15 different ligand complexes with tryptophan synthase at 1.7 to 2.4 A resolution. Despite these successes, significant chemical questions remain as the resolution of these structures does not allow for a detailed chemical mechanism to be established for the substrate transformation. Yet chemical level details such as protonation and hybridization states are critical for understanding enzymatic mechanism and function. Even under moderately high resolution, these are difficult to determine from X-ray crystallography alone. The chemical shift in nuclear magnetic resonance (NMR), however, is an extremely sensitive probe of chemical environment, making solid- state NMR and X-ray crystallography a powerful combination for defining chemically-detailed three dimensional structures. Here we adopt a combined X-ray crystallography/solid state NMR/ab initio calculation approach to determine the chemically-rich crystal structures of several key intermediates in the multistep transformation of substrate to product in the 2-subunit of tryptophan synthase. Models of the active site are developed using a synergistic approach in which the structure of this reactive substrate/analogue is freely optimized using computational chemistry in the presence of side chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues can be uniquely distinguished by their calculated effect on the chemical shifts, measured at specifically 13C and 15N-labeled positions on substrates/analogues, coenzyme and site catalytic residues. This treatment provides an accurate chemically-detailed starting point for dynamics and reaction coordinate scans that have already provided unique insight into the connection between chemical structure and the resulting local electrostatic fields that help drive and direct the next step in the catalysis.
The vitamin-B6-dependent tryptophan synthase complex catalyses the last two steps in the synthesis of L-Trp. Tryptophan synthase homologues are found in bacteria, yeasts, molds, plants, and some protozoans. The absence of a synthetic pathway for L-Trp in higher animals and in humans makes the tryptophan synthase nanomachine a potential target both for the development of herbicides, and for the design of drugs to treat infectious disease. Consequently, understanding the catalytic mechanism could provide useful insights for developing tryptophan synthase as an important target for drug design, or for the development of herbicides.
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