Substrate Stereoselectivity of Human Indoleamine 2,3-Dioxygenase

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Indoleamine 2,3-dioxygenase (IDO) and Tryptophan 2,3-dioxygenase (TDO) are two heme-containing enzymes that catalyze the oxidative cleavage of tryptophan (Trp) to N-formyl-kynurenine, the initial and rate-limiting step of the kynurenine pathway. Although IDO and TDO catalyze the same reaction, they exhibit distinct structural and functional features. TDO plays an important role in regulating homeostatic serum Trp concentrations, whereas IDO is involved in a wide spectrum of immune related pathophysiologies. It has been shown that immune cells express IDO to suppress pathogen growth by depleting the local Trp concentration and by producing cytotoxic metabolites. Ironically, IDO produced in the placenta and by cancer cells has also been implicated in inhibiting the proliferation of immune cells by similar mechanisms. To study the substrate-protein interaction in human IDO (hIDO), as compared to human TDO (hTDO), we have constructed and studied three mutants of hIDO, including S167H and F226Y (in which the two critical amino acids in the active site were mutated to mimic TDO), as well as trIDO (in which the N-terminal domain absent in TDO was truncated). The structural and enzymatic properties of each mutant were systematically examined with optical absorption and resonance Raman spectroscopies. The data were evaluated against the wild type hIDO and hTDO. It was concluded that: (1) the mutation of F226 to Tyr changes the substrate stereoselectivity to be “TDO-like”; (2) the mutation of S167 to His causes the inactivation of IDO; and (3) the N-terminal domain of IDO is critical for Trp binding and activity. These studies will be discussed in the context of the dioxygen chemistry carried out by these two important heme-containing enzymes.