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Indoleamine 2,3‐dioxygenase (IDO) is a monomeric heme enzyme that catalyzes the oxidative cleavage of tryptophan (L‐Trp) to form N‐formyl‐kynurenine. Similar to other heme proteins, IDO only binds to O2 when the heme iron is ferrous (FeII), thereby rendering the enzyme active. Thus, ascorbate (Asc, a reducing agent) and methylene blue (MB, an electron carrier) are commonly added to in vitro IDO assay systems. However, Asc and MB have been recently reported to significantly impact the measurement of the enzymatic parameters of vertebrate IDO. Aspergillus fumigatus is a filamentous fungus and the most common cause of invasive aspergillosis; it has three IDO genes (IDOα, IDOβ, and IDOγ). The FeII–O2 IDOs of A. fumigatus, particularly FeII–O2 IDOγ, have relatively long half‐lives in their autoxidation; however, the autoxidation was accelerated by Asc. Similar to vertebrate IDOs, Asc acted as a competitive (or mixed‐competitive) inhibitor of the IDOs of A. fumigatus. A positive correlation (in the order of IDOγ > IDOβ > IDOα) was observed between the inhibitory sensitivity of the IDOs to Asc and the facilitation of their autoxidation by Asc. The FeII–O2 IDO can repeat the dioxygenase reaction as long as it reacts with L‐Trp; however, substrate‐free FeII–O2 IDO is converted into inactive FeIII–IDO by autoxidation. Thus, L‐Trp (which keeps the IDO active) competes with Asc (which inactivates IDO by accelerating autoxidation). This is probably why Asc, which is structurally quite different from L‐Trp, appears to function as a competitive (or mixed‐competitive) inhibitor of IDOs. [ABSTRACT FROM AUTHOR]

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyse the same reaction, oxidative cleavage of L-tryptophan (L-Trp) to N -formyl-kynurenine. In both enzymes, the ferric form is inactive and ascorbate (Asc) is frequently used as a reductant in in vitro assays to activate the enzymes by reducing the heme iron. Recently, it has been reported that Asc activates IDO2 by acting as a reductant; however, it is also a competitive inhibitor of the enzyme. Here, the effect of Asc on human TDO (hTDO) is investigated. Similar to its interaction with IDO2, Asc acts as both a reductant and a competitive inhibitor of hTDO in the absence of catalase, and its inhibitory effect was enhanced by the addition of H2O2. Interestingly, however, no inhibitory effect of Asc was observed in the presence of catalase. TDO is known to be activated by H2O2 and a ferryl-oxo (FeIV=O) intermediate (Compound II) is generated during the activation process. The observation that Asc acts as a competitive inhibitor of hTDO only in the absence of catalase can be explained by assuming that the target of Asc is Compound II. Asc seems to compete with L-Trp in an unusual manner. [ABSTRACT FROM AUTHOR]

Calcyphosine is an EF-hand Ca2+-binding protein, which was first isolated from the canine thyroid. It is phosphorylated in a cyclic AMP (cAMP)-dependent manner; then it is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. Here, we isolated the DNA complementary to RNA (cDNA) of an EF-hand Ca2+-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca2+-binding protein showed significant similarity (about 40% identity) with those of mammal calcyphosines, and the intron positions were well conserved between the sponge and human calcyphosine genes. We considered that the isolated cDNA was that of sponge calcyphosine, and that sponge and mammalian calcyphosines evolved from a common ancestor gene. Recent cDNA projects have revealed that a calcyphosine cDNA is also expressed by human, mouse, and the ascidia. These cDNAs have more than 60% identity with sponge calcyphosine and each other, and all are composed of 208 amino acid residues. On the constructed phylogenetic trees, calcyphosines are essentially divided into two groups, types-I and -II calcyphosines. Type-I calcyphosine may be specific to mammals, and type-II is widely distributed among metazoan species. This suggests that type-II calcyphosine is a rather ancient gene with some essential function. [Copyright &y& Elsevier]