Analysis of Reaction Intermediates in Tryptophan 2,3-Dioxygenase: A Comparison with Indoleamine 2,3-Dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme-containing enzymes that catalyze the O2-dependent oxidation of l-tryptophan (l-Trp) in biological systems. Although many decades have passed since their discovery, the mechanism of tryptophan oxidation has not been established. It has been widely assumed that IDO and TDO react using the same mechanism, although there is no evidence that they do. For IDO, a Compound II (ferryl) species accumulates in the steady state and is implicated in the mechanism; in TDO, no such species has ever been observed. In this paper, we examine the kinetics of tryptophan oxidation in TDO. We find no evidence for the accumulation of Compound II during TDO catalysis. Instead, a ternary [Fe(II)-O2, l-Trp] complex is detected under steady state conditions. The absence of a Compound II species in the steady state in TDO is not due to an intrinsic inability of the TDO enzyme to form ferryl heme, because Compound II can be formed directly through a different route in which ferrous heme is reacted with peroxide. We interpret the data to mean that the rate-limiting step in the IDO and TDO mechanisms is not the same.

Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM.

The kynurenine pathway is the major route of L-tryptophan (L-Trp) catabolism in biology, leading ultimately to the formation of NAD(+). The initial and rate-limiting step of the kynurenine pathway involves oxidation of L-Trp to N-formylkynurenine. This is an O2-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237-244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of L-Trp, 1-methyl-L-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.

Binding of l-kynurenine to X. campestris tryptophan 2,3-dioxygenase.

The kynurenine pathway is the major route of tryptophan metabolism. The first step of this pathway is catalysed by one of two heme-dependent dioxygenase enzymes - tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) - leading initially to the formation of N-formylkynurenine (NFK). In this paper, we present a crystal structure of a bacterial TDO from X. campestris in complex with l-kynurenine, the hydrolysed product of NFK. l-kynurenine is bound at the active site in a similar location to the substrate (l-Trp). Hydrogen bonding interactions with Arg117 and the heme 7-propionate anchor the l-kynurenine molecule into the pocket. A mechanism for the hydrolysis of NFK in the active site is presented.