Mechanistic Studies of Tryptophan Oxidizing Enzymes

Georgia State University Research Foundation, Inc., Atlanta GA

Investigators

Abstract

Heme iron active sites are present in a wide variety of proteins and enzymes. The different biological functions they perform include oxygen binding, storage, transfer, and activation for hydroxylation, peroxidation, and desaturation. This project examines the molecular biochemistry exhibited by two structurally diverse heme-containing enzymes that oxidize either free tryptophan or protein-bound tryptophan residues. Tryptophan 2,3-dioxygenase (TDO) inserts two oxygen atoms into free tryptophan in a four electron oxidizing process by a b-type heme cofactor. This enzyme represents a potentially new hemoprotein dioxygenase superfamily whose oxygenase activity remains poorly understood. MauG is a novel enzyme that utilizes two c-type hemes to catalyze a posttranslational modification of a 119 KDa protein. Such a modification endows endogenous tryptophan residues with a new catalytic activity. The MauG-catalyzed reaction is a six-electron oxidation process and the utilization of two c-type hemes to perform a hydroxylation reaction and the subsequent oxidation reactions are unprecedented. Novel high valent iron intermediates have been trapped from these tryptophan oxidizing enzymes. This research will establish a better definition of the requirements needed to attain the unusual Fe(IV) intermediates as well as elucidate their relation to oxidizing the free or protein-bound tryptophan substrates. A biochemical and spectroscopic approach employing site-directed mutagenesis, electron paramagnetic resonance (EPR) spectroscopy, Mössbauer spectroscopy, mass spectrometric analysis, and structural determination as well as quantum mechanics computational methods will be utilized to study the electronic structure of the intermediates in these enzymes. These studies are directed toward obtaining detailed insight into electronic and geometric structural contributions to the formation and stabilization of high valent iron intermediates, the electronic structure of reactive oxygen intermediates, and the heme-dependent tryptophan oxidizing mechanisms contrasts to non-heme catalysis. Broader Impacts: This research will provide the opportunity for motivated students, including underrepresented populations, to participate in the frontier of molecular biochemistry research. Students exposure to an array of biochemical and biophysical techniques will be integrated with hands-on training. A video tutorial of electron paramagnetic resonance (EPR) spectroscopy will be developed during the experimental process to provide a thorough demonstration of EPR-centered techniques in studying the chemical properties of important biomolecular systems.