Substrate-protein interaction in human tryptophan dioxygenase

Abstract

Tryptophan 2, 3-dioxygenase (hTDO) and indoleamine 2, 3-dioxygenase (hIDO) are the only two classes of heme-containing dioxygenases in humans, both of which catalyze the oxidative degradation of the L-Trp to N-formyl kynurenine. Although the two enzymes catalyze the same reaction, and show high structural homology, they are engaged in distinct physiological functions and show distinct biochemical properties. The expression of hIDO is induced by IFN-gamma and role of hIDO has been implicated in diverse range of pathophysiological conditions, whereas hTDO is induced by glucocortocoid hormones and deals with the systemic regulation of the Trp flux in the body. Hence understanding the differences between hTDO and hIDO offer invaluable structural information for the design of new inhibitors selective for hIDO. I have characterized hTDO for the first time using resonance Raman spectroscopy and optical absorption spectroscopies. Through my data I have demonstrated that the distal pocket of the two heme enzymes in presence of L-Trp are distinctly different from each other. I have proposed that the initial deprotonation of the indole NH of Trp in hTDO is carried by the evolutionary conserved distal His, whereas in hIDO it is believed that the heme-bound dioxygen performs this deprotonation. To test the proposed model, I have mutated this conserved His to demonstrate its critical role in the oxygen reaction of hTDO. Furthermore, I have performed solvent isotope effect experiments as a function of pH to elucidate the role of deprotonation in the oxygen reaction mechanism. I have also used different Trp analogues to determine the substrate ligand interactions in the active site of hTDO by using resonance Raman spectroscopy. Finally, I have identified the stable ternary complex of hTDO and studied the kinetics of the oxygen reaction by using the stopped flow module. I have established for the first time the vibrational characteristics of the oxygen bound heme moiety of hTDO by directly observing the Fe-O2 stretching mode of hTDO by our continuous flow resonance Raman system. Overall, I observed that the evolutionary conserved distal His plays an important role in oxygen binding and overall stability of the ternary complex of hTDO.