Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygenbinding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.cancer ͉ heme enzymes ͉ immunomodulation ͉ indoleamine 2,3-dioxygenase T ryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze the oxidative cleavage of the L-tryptophan (L-Trp) pyrrole ring, the first and rate-limiting step in L-Trp catabolism through the kynurenine pathway (1-3). In addition, IDO has been implicated in a diverse range of physiological and pathological conditions, including suppression of T cell proliferation, maternal tolerance to allogenic fetus, and immune escape of cancers (4-8), and is an attractive target for drug discovery against cancer and autoimmune and other diseases (2, 9-12).Despite catalyzing identical biochemical reactions (Fig. 1a), the sequence similarity between TDO and IDO is extremely low. An alignment of their sequences is only possible based on their structures, which suggests a sequence identity of 10% between them (Fig. 1b). In comparison, Xanthomonas campestris TDO shares 34% sequence identity with human TDO (Fig. 1b), demonstrating the remarkable evolutionary conservation of this enzyme. TDO is a homotetrameric enzyme and is highly specific for L-Trp and related derivatives such as 6-fluoro-Trp as the substrate. In comparison, IDO is monomeric, and shows activity toward a larger collection of substrates, including L-Trp, Dtryptophan (D-Trp), serotonin, and tryptamine (3), although the K m for D-Trp is Ϸ100-fold higher than that for L-Trp (13). The structure of human IDO in the catalytically inactive, ferric [Fe(III)]-heme state in complex with the 4-phenylimidazole inhibitor has recently been reported (14). Although this structure gave information about important active site residues, the inhibitor is coordinat...

Bacterial toxin-antitoxin (TA) systems serve a variety of physiological functions including regulation of cell growth and maintenance of foreign genetic elements. Sequence analyses suggest that TA families are linked by complex evolutionary relationships reflecting likely swapping of functional domains between different TA families. Our crystal structures of Phd-Doc from bacteriophage P1, the HigA antitoxin from Escherichia coli CFT073, and YeeU of the YeeUWV systems from E. coli K12 and Shigella flexneri confirm this inference and reveal additional, unanticipated structural relationships. The growth-regulating Doc toxin exhibits structural similarity to secreted virulence factors that are toxic for eukaryotic target cells. The Phd antitoxin possesses the same fold as both the YefM and NE2111 antitoxins that inhibit structurally unrelated toxins. YeeU, which has an antitoxin-like activity that represses toxin expression, is structurally similar to the ribosome-interacting toxins YoeB and RelE. These observations suggest extensive functional exchanges have occurred between TA systems during bacterial evolution.