Structure and Function of Heme-based Dioxygenases

SUMMARY Tryptophan (Trp) is the least abundant essential amino acid. The majority of our dietary Trp is metabolized through the kynurenine (KYN) pathway. The first and rate-limiting step of the KYN pathway is catalyzed by three heme-based dioxygenases, tryptophan dioxygenase (hTDO), indoleamine 2,3-dioxygenase 1 (hIDO1), and indoleamine 2,3-dioxygenase 2 (hIDO2). Recently it was found that the three dioxygenases are expressed in cancer cells to promote cancer immune escape. Consequently they have been considered as key drug targets for cancer immunotherapy. Despite their importance, the structural and functional properties of these enzymes remain elusive, which has hindered the progress of the field. The central hypothesis of this project, as supported by our preliminary data, is (i) the functional properties of the three dioxygenases are regulated by cellular metabolites and (ii) each dioxygenase exhibits distinct structural features and possesses unique drug binding sites. We will test our hypothesis by addressing two specific aims: (i) identify cellular metabolites that interact with each dioxygenase and define the related regulatory mechanisms, and (ii) define structural differences between the three dioxygenases and determine new small molecule binding sites in each dioxygenase. We will use a new high- throughput mass spectrometry-based screening technology to identify metabolites that interact with each dioxygenase and use X-ray crystallography and spectroscopic techniques to define their specific molecular interactions and functional consequences. These studies will reveal previously unknown cellular players in dioxygenase-related human physiology that may impact the specific functions of these enzymes in cancer and other diseases, thereby offering novel information enabling innovative molecular approaches for disease prevention and control. In parallel, we will use an integrated approach, involving a wide spectrum of biochemical and biophysical techniques, and a group of structurally diverse inhibitors as probes to define unique structural features and new small molecule binding sites in each dioxygenase. The outcome of these studies will offer important knowledge enabling better understanding of structure-and-function relationships of the three heme- based dioxygenases and expanding our toolkit for rational design of enzyme-selective inhibitors. We have assembled a team of experts to carry out this innovative project with the multifaceted approach. These studies will address significant gaps in our knowledge of molecular mechanisms underlying the biological functions of the three dioxygenases and provide important new insights into related drug development and disease treatment.