Unusual Evolution of Cephalopod Tryptophan Indole-Lyases.

Tryptophan indole-lyase (TIL), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the hydrolysis of L-tryptophan (L-Trp) to indole and ammonium pyruvate. TIL is widely distributed among bacteria and bacterial TILs consist of a D2-symmetric homotetramer. On the other hand, TIL genes are also present in several metazoans. Cephalopods have two TILs, TILα and TILß, which are believed to be derived from a gene duplication that occurred before octopus and squid diverged. However, both TILα and TILß individually contain disruptive amino acid substitutions for TIL activity, and neither was active when expressed alone. When TILα and TILß were coexpressed, however, they formed a heterotetramer that exhibited low TIL activity. The loss of TIL activity of the heterotetramer following site-directed mutagenesis strongly suggests that the active heterotetramer contains the TILα/TILß heterodimer. Metazoan TILs generally have lower kcat values for L-Trp than those of bacterial TILs, but such low TIL activity may be rather suitable for metazoan physiology, where L-Trp is in high demand. Therefore, reduced activity may have been a less likely target for purifying selection in the evolution of cephalopod TILs. Meanwhile, the unusual evolution of cephalopod TILs may indicate the difficulty of post-gene duplication evolution of enzymes with catalytic sites contributed by multiple subunits, such as TIL.

A single amino acid residue regulates the substrate affinity and specificity of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyses the oxidative cleavage of L-Trp. The ciliate Blepharisma stoltei has four IDO genes (IDO-I, -II, -III and -IV), which seem to have evolved via two sequential gene duplication events. Each IDO enzyme has a distinct enzymatic property, where IDO-III has a high affinity for L-Trp, whereas the affinity of the other three isoforms for L-Trp is low. IDO-I also exhibits a significant catalytic activity with another indole compound: 5-hydroxy-l-tryptophan (5-HTP). IDO-I is considered to be an enzyme that is involved in the biosynthesis of the 5-HTP-derived mating pheromone, gamone 2. By analysing a series of chimeric enzymes based on extant and predicted ancestral enzymes, we identified Asn131 in IDO-I and Glu132 in IDO-III as the key residues responsible for their high affinity for each specific substrate. These two residues were aligned in an identical position as the substrate-determining residue (SDR). Thus, the substrate affinity and specificity are regulated mostly by a single amino acid residue in the Blepharisma IDO-I and IDO-III enzymes.

Efficient tryptophan-catabolizing activity is consistently conserved through evolution of TDO enzymes, but not IDO enzymes.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) enzymes have independently evolved to catalyze the first step in the catabolism of tryptophan (L-Trp) through the kynurenine pathway. TDO is found in almost all metazoan and many bacterial species, but not in fungi. We show that TDO enzymes have high catalytic-efficiency for L-Trp catabolism, regardless of their biological origin, suggesting that TDO has been an L-Trp-specific degrading enzyme throughout its evolution. Meanwhile, IDO was initially discovered in mammals, and subsequently has been found in lower vertebrates, several invertebrates, fungi and a number of bacterial species. Some lineages have independently generated multiple IDO paralogues through gene duplications. Interestingly, only mammalian IDO1s and fungal "typical" IDOs have high affinity and catalytic efficiency for L-Trp catabolism, comparable to TDOs. We show that invertebrate IDO enzymes have low affinity and catalytic efficiency for L-Trp catabolism. We suggest that the phylogenetic distribution of "low catalytic-efficiency IDOs" indicates the ancestral IDO also had low affinity and catalytic efficiency for L-Trp catabolism. IDOs with high catalytic-efficiency for L-Trp-catabolism may have evolved in certain lineages to fulfill particular biological roles. The low catalytic-efficiency IDOs have been well conserved in a number of lineages throughout their evolution, although it is not clear that the enzymes contribute significantly to L-Trp catabolism in these species. Investigation of other substrates and functions of the ancestral IDO and low catalytic efficiency IDOs may identify additional biological roles for these enzymes.

Human indoleamine 2,3-dioxygenase-2 has substrate specificity and inhibition characteristics distinct from those of indoleamine 2,3-dioxygenase-1.

Indoleamine 2,3-dioxygenase-2 (IDO2) is one of three enzymes (alongside tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (IDO1)) that catalyse dioxygenation of L-tryptophan as the first step in the kynurenine pathway. Despite the reported expression of IDO2 in tumours, some fundamental characteristics of the enzyme, such as substrate specificity and inhibition selectivity, are still to be clearly defined. In this study, we report the kinetic and inhibition characteristics of recombinant human IDO2. Choosing from a series of likely IDO2 substrates, we screened 54 tryptophan derivatives and tryptophan-like molecules, and characterised the 8 with which the enzyme was most active. Specificity of IDO2 for the two isomers of 1-methyltryptophan was also evaluated and the findings compared with those obtained in other studies on IDO2 and IDO1. Interestingly, IDO2 demonstrates behaviour distinct from that of IDO1 in terms of substrate specificity and affinity, such that we have identified tryptophan derivatives that are mutually exclusive as substrates for IDO1 and IDO2. Our results support the idea that the antitumour activity of 1-Me-D-Trp is unlikely to be related with competitive inhibition of IDO2, and also imply that there are subtle differences in active site structure in the two enzymes that may be exploited in the development of specific inhibitors of these enzymes, a route which may prove important in defining their role(s) in cancer.

Indoleamine 2,3-dioxygenases with very low catalytic activity are well conserved across kingdoms: IDOs of Basidiomycota.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme and is found in animals, fungi and bacteria. In fungi, its primary role is to supply nicotinamide adenine dinucleotide (NAD(+)) via the kynurenine pathway. A number of organisms possess more than one IDO gene, for example, mammals have IDO1 and IDO2 genes. We previously reported that the Pezizomycotina fungi commonly possess three types of IDO genes, IDOα, IDOß and IDOγ. In this study, we surveyed the nature of IDO genes from Basidiomycota fungi, which are categorized into three subphyla (Agaricomycotina, Pucciniomycotina and Ustilaginomycotina). The Agaricomycotina fungi generally have three types of IDO genes (IDOa, IDOb and IDOc), which are distinct from Pezizomycotina three isozymes. Pucciniomycotina and Ustilaginomycotina species possess two types of IDO; one forms a monophyletic clade with Agaricomycotina IDOs in the phylogenetic tree, these IDOs are referred to as "typical Basidiomycota IDOs". The other is IDOγ, which showed more than 40% identity with Pezizomycotina and ciliate IDOγ. We previously demonstrated that IDO2 in mammals and IDOγ in Perzizomycotina fungi have much lower catalytic efficiencies in an in vitro assay, compared with the other IDO isoforms found in the respective species. We have developed a functional assay to determine whether particular IDO enzymes have sufficient enzymatic activity to rescue a yeast strain where IDO-deletion has rendered it auxotrophic for nicotinic acid. IDOα and IDOß showed comparable catalytic efficiency, both of them could function in the Pezizomycotina fungal L-Trp metabolism. The catalytic efficiency and functional capacity of the Basidiomycota IDOa and IDOb were similar to Pezizomycotina IDOα/IDOß. We found that Basidiomycota IDOc could not rescue the nicotinic acid auxotroph, similar to other IDO enzymes with low catalytic efficiency (mammalian IDO2 and most fungal IDOγ). Our study suggests that some fungal IDO enzymes function in tryptophan metabolism and NAD(+) supply. In contrast, other IDO enzymes do not possess sufficient Trp-metabolising capacity to supply NAD(+). Although the role of these low catalytic efficiency IDOs is not clear, it is interesting to note that IDO enzymes possessing these characteristics have evolved across different kingdoms.

Metazoan tryptophan indole-lyase: Are they still active?

Tryptophan indole-lyase (TIL), also known as tryptophanase, is a pyridoxal-5'-phosphate dependent bacterial enzyme that catalyzes the reversible hydrolytic cleavage of l-tryptophan (l-Trp) to indole and ammonium pyruvate. TIL is also found in some metazoans, and they may have been acquired by horizontal gene transfer. In this study, two metazoans, Nematostella vectensis (starlet sea anemone) and Bradysia coprophila (fungus gnat) TILs were bacterially expressed and characterized. The kcat values of metazoan TILs were low, < 1/200 of the kcat of Escherichia coli TIL. By contrast, metazoan TILs showed lower Km values than the TILs of common bacteria, indicating that their affinity for l-Trp is higher than that of bacterial TILs. Analysis of a series of chimeric enzymes based on B. coprophila and bacterial TILs revealed that the low Km value of B. coprophila TIL is not accidental due to the substitution of a single residue, but is due to the cooperative effect of multiple residues. This suggests that high affinity for l-Trp was positively selected during the molecular evolution of metazoan TIL. This is the first report that metazoan TILs have low but obvious activity.

Inhibitory effect of ascorbate on tryptophan 2,3-dioxygenase.

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.

Molecular evolution and characterization of fungal indoleamine 2,3-dioxygenases.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes. Mammalian IDO expression is induced by cytokines and has antimicrobial and immunomodulatory effects. A major role of mammalian TDO is to supply nicotinamide adenine dinucleotide (NAD(+)). In fungi, the IDO homologue is thought to be expressed constitutively and supply NAD(+), as TDO is absent from their genomes. Here, we reveal the distribution of IDO genes among fungal species and characterize their enzymatic activity. The yeast, Saccharomyces cerevisiae has only one IDO gene, whereas the koji-mold, Aspergillus oryzae has two genes, IDOα and IDOß. The A. oryzae IDOα showed more similar enzymatic properties to those of S. cerevisiae IDO than IDOß, suggesting that the A. oryzae IDOα is a functional homologue of the S. cerevisiae IDO. From the IDOß gene, two isoforms, IDOß and IDOß(+) could be generated by alternative splicing. The latter contained a 17 amino acids insertion which were encoded by the first intron of IDOß gene. In comparison to IDOß(+), bacterially expressed IDOß showed much lower K(m) value and more than five-times faster V(max) value, resulting in 85 times higher catalytic efficiency; i.e., the removal of the domain encoded by the first intron from IDOß(+) increases its enzymatic activity drastically. This might be a unique regulation mechanism of the L-Trp metabolism in the A. oryzae. The levo-1-methyl tryptophan (L-1MT) is a good inhibitor of both IDO1 and IDO2. However, the activity of fungal IDOs tested was not inhibited at all by L-1MT.

Methylene blue and ascorbate interfere with the accurate determination of the kinetic properties of IDO2.

Indoleamine 2,3-dioxygenases (IDOs) catalyze the oxidative cleavage of L-tryptophan (Trp) to N-formylkynurenine. Two IDOs, IDO1 and IDO2, are present in vertebrates. IDO1 is a high-affinity Trp-degrading enzyme involved in several physiological processes. By comparison, IDO2 generally has been reported to have low affinity (high Km -value) for Trp, and the enzyme's in vivo function remains unclear. Using IDOs from different species, we show that compared with ferrous-oxy (Fe2+ -O2 ) IDO1, Fe2+ -O2 IDO2 is substantially more stable and engages in multiple turnovers of the reaction in the absence of a reductant. Without reductant, Fe2+ -O2 IDO2 showed Km -values in the range of 80-356 µM, that is, values substantially lower than reported previously and close to the physiological concentrations of Trp. Methylene blue and ascorbate (Asc), used commonly as the reducing system for IDO activity determination, significantly affected the enzymatic activity of IDO2: In combination, the two reductants increased the apparent Km - and kcat -values 8- to 117-fold and 2-fold, respectively. Asc alone both activated and inhibited IDO2 by acting as a source of electrons and as a weak competitive inhibitor, respectively. In addition, ferric (Fe3+ ) IDO1 and IDO2 exhibited weak dioxygenase activity, similar to tryptophan 2,3-dioxygenase. Our results shed new light in the enzymatic activity of IDO2, and they support the view that this isoform of IDO also participates in the metabolism of Trp in vivo.

A comprehensive comparison of the metazoan tryptophan degrading enzymes.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) have an independent origin; however, they have distinctly evolved to catalyze the same reaction. In general, TDO is a single-copy gene in each metazoan species, and TDO enzymes demonstrate similar enzyme activity regardless of their biological origin. In contrast, multiple IDO paralogues are observed in many species, and they display various enzymatic properties. Similar to vertebrate IDO2, invertebrate IDOs generally show low affinity/catalytic efficiency for L-Trp. Meanwhile, two IDO isoforms from scallop (IDO-I and -III) and sponge IDOs show high L-Trp catalytic activity, which is comparable to vertebrate IDO1. Site-directed mutagenesis experiments have revealed that primarily two residues, Tyr located at the 2nd residue on the F-helix (F2nd) and His located at the 9th residue on the G-helix (G9th), are crucial for the high affinity/catalytic efficiency of these 'high performance' invertebrate IDOs. Conversely, those two amino acid substitutions (F2nd/Tyr and G9th/His) resulted in high affinity and catalytic activity in other molluscan 'low performance' IDOs. In human IDO1, G9th is Ser167, whereas the counterpart residue of G9th in human TDO is His76. Previous studies have shown that Ser167 could not be substituted by His because the human IDO1 Ser167His variant showed significantly low catalytic activity. However, this may be specific for human IDO1 because G9th/His was demonstrated to be very effective in increasing the L-Trp affinity even in vertebrate IDOs. Therefore, these findings indicate that the active sites of TDO and IDO are more similar to each other than previously expected.

Duodenal adenocarcinoma successfully diagnosed with transabdominal ultrasonography.

Adenocarcinoma arising from the duodenum is relatively rare. Diagnosis of this disease at an early stage is difficult because its symptoms are usually nonspecific. We herein present a case in which duodenal adenocarcinoma was successfully found by transabdominal ultrasonography. Under ultrasonography, the tumor was located in the proximal duodenum apart from the papilla of Vater, and the serosa was intact. Other diagnostic modalities showed no evidence of adjacent organ invasion or distant metastasis. Therefore, pancreatoduodenectomy was performed and the postoperative course was uneventful. The ultrasonographic findings corresponded well with the pathological diagnosis. The following three procedures were essential in this case: systematic scanning of the digestive tract to determine the location of the lesion, graded compression ultrasound to remove air bubbles from the region of interest, and precise observation of the intestinal walls using proper transducers. The precise and skillful performance of transabdominal ultrasonography using a suitable device can help to diagnose duodenal adenocarcinoma, a rare malignancy.

Bacterial expression and characterization of molluscan IDO-like myoglobin.

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic alpha- and beta-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His(74), His(288), and His(332) were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His(332) is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His(74) and His(288) might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.

Novel Specificity of IDO Enzyme Involved in the Biosynthesis of Mating Pheromone in the Ciliate Blepharisma stoltei.

Mating pheromones (gamone 1 and gamone 2) in the ciliate Blepharisma are biologically active substances that trigger sexual reproduction (conjugation) under starvation conditions. Gamone 1 is a glycoprotein secreted by type I cells, and gamone 2 is a tryptophan (Trp)-derivative compound secreted by type II cells. Both gamones stimulate complementary mating type cells to promote each gamone production and induce pair formation. To elucidate the biosynthetic pathway of gamone 2, we investigated the enzymes involved in the pathway and the specificity of the enzymes. An RNA-seq analysis revealed that Blepharisma stoltei (Heterotrichea) possesses four indoleamine 2,3-dioxygenase (IDO) genes showing distinct expression patterns. Along with results from real-time PCR, these findings demonstrated that each IDO gene has different expression patterns that depend on the cellular conditions. Expression of IDO-I was correlated with the intensity of gamone 2 expression, and the recombinant IDO-I protein showed catalytic activity for 5-hydroxy-L-Trp (5-HTP) but very weak activity for L-Trp. Our results indicate that IDO-I is an enzyme evolutionary specialized to gamone 2 production in Blepharisma, and that the biosynthetic pathway for gamone 2 uses 5-HTP as an intermediate.

High l-Trp affinity of indoleamine 2,3-dioxygenase 1 is attributed to two residues located in the distal heme pocket.

Indoleamine 2, 3-dioxygenase (IDO) catalyzes the oxidative cleavage of the pyrrole ring of l-Trp to generate N-formyl-kynurenine. Two IDO genes, IDO1 and IDO2, are found in vertebrates. Mammalian IDO1s are high-affinity, l-Trp-degrading enzymes, whereas IDO2s generally have a relatively low affinity. It has been suggested that the distal-Ser (corresponding to Ser167 of human IDO1) was crucial for improvement in the affinity for l-Trp but this idea was insufficient to explain the high affinity shown by mammalian IDO1. In this study, the amino acid sequences of vertebrate ancestral IDO1 and ancestral IDO2 were inferred, and bacterially expressed ancestral IDOs were characterized. Although the amino acid sequences of the enzymes shared high identity (86%) with each other, they showed distinct enzymatic properties. In analyses of a series of ancestral IDO1/IDO2 chimeric enzymes and their variants, the distal-Tyr (corresponding to Tyr126 of human IDO1) was detected as another and was probably the most crucial residue for high l-Trp affinity. The two amino acid substitutions (distal-Ser to Thr and distal-Tyr to His) drastically decreased the l-Trp affinity and catalytic efficiency of IDO1s. Conversely, two substitutions (distal-Thr to Ser and distal-His to Tyr) were sufficient to bestow IDO1-like high affinity on ancestral and chicken IDO2.

Do molluscs possess indoleamine 2,3-dioxygenase?

Indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor, and it has since been isolated from several archaegastropods. The amino acid sequences and genomic structures of IDO-like Mbs show significant homology with those of mammalian IDOs, suggesting that they have evolved from a common ancestral gene. However, details of the evolutionary relationships between them remain unknown. Here, we isolated a novel multicopy gene from Sulculus named molluscan IDO-like protein (MIP). The amino acid sequences of MIPs show the highest homology (about 60% identity) with Sulculus IDO-like Mb, and their exon/intron structures are also highly homologous. However, MIPs are mainly expressed in the gut whereas IDO-like Mb was found only in the buccal mass, suggesting that MIPs are not simply isoforms of IDO-like Mb. A bacterial expression study showed that MIP is a heme-binding protein, and that His335 is the proximal ligand of heme. Although we could not detect IDO activity using a recombinant glutathione S-transferase (GST)-MIP fusion protein in the present study, MIP should have some function other than that of an oxygen carrier like myoglobin, and it might in fact be molluscan IDO.

Low efficiency IDO2 enzymes are conserved in lower vertebrates, whereas higher efficiency IDO1 enzymes are dispensable.

Indoleamine 2,3-dioxygenase (IDO) is a Trp-degrading enzyme that catalyzes the first step in the kynurenine pathway. Two IDO genes, IDO1 and IDO2, are found in vertebrates and the timing of the gene duplication giving rise to the genes has been controversial. In the present study, we report that several fishes and two turtles also have both IDO1 and IDO2. This represents definitive evidence for the gene duplication occurring before the divergence of vertebrates, with IDO1 having been lost in a number of lower vertebrate lineages. IDO2 enzymes have a relatively low affinity for l-Trp; however, Anolis carolinensis (lizard) IDO2 has an affinity for l-Trp comparable to mammalian IDO1 enzymes. We identified a Ser residue located in the distal heme pocket of IDO1 (distal-Ser) (corresponding to Ser167 of human IDO1) that is conserved in all IDO1 enzymes and the lizard IDO2. This residue is conserved as Thr (distal-Thr) in other IDO2 enzymes. Biochemical analyses, using IDO variants with either Ser or Thr substitutions, suggest that the distal-Ser change was crucial for the improvement in affinity for l-Trp in ancient IDO1. The ancestral IDO1 likely had a 'moderate' enzymatic efficiency for l-Trp, clearly higher than IDO2 but lower than mammalian IDO1. The distal-Ser of lizard IDO2 bestows a high affinity for l-Trp, however, this unique IDO2 has a low enzymatic efficiency because of its very low catalytic velocity. Thus, low efficiency IDO2 enzymes have been conserved throughout vertebrate evolution, whereas higher efficiency IDO1 enzymes are dispensable in many lower vertebrate lineages.

Genomic structure of the sponge, Halichondria okadai calcyphosine gene.

Calcyphosine is an EF-hand Ca(2+)-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 Ca(2+)-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca(2+)-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.

Comparison of the sequences of Turbo and Sulculus indoleamine dioxygenase-like myoglobin genes.

Some archaeogastropodic molluscs, including Sulculus and Turbo, contain an unusual approximately 40 kDa myoglobin in their buccal masses. This myoglobin can bind oxygen reversibly, but has a lower oxygen affinity than vertebrate and invertebrate myoglobins. Amino acid sequences clearly show that Sulculus and Turbo myoglobins evolved not from the globin gene but from the gene for indoleamine dioxygenase (IDO), a tryptophan-degrading enzyme. The Turbo myoglobin gene has been determined to consist of 14 exons and 13 introns. Compared with the known Sulculus IDO-like myoglobin gene, all splice junctions except two are conserved exactly between the two genes. The exon/intron organization of these myoglobin genes is also highly homologous with human IDO (ten exon/nine intron structure); splice junctions of six introns were exactly conserved among the three genes, suggesting that these introns have been conserved for at least 600 million years. To look for putative IDO genes in Turbo or Sulculus, we re-examined the genomic DNA fragments amplified by PCR in full detail, and found intron 2 in two distinct Sulculus fragments (A and B). Fragment A with a 576 bp intron corresponded exactly to the myoglobin gene of Sulculus. On the other hand, fragment B, containing a 239 bp intron, differed significantly from fragment A in nucleotide and translated amino acid sequences. Detailed sequence comparison suggests that fragment B may be derived from a putative IDO gene of Sulculus.

The structural organization of ascidian Halocynthia roretzi troponin I genes.

The organization of troponin I (TnI) genes from the ascidian Halocynthia roretzi have been determined. Halocynthia possesses roughly two types of TnI isoforms. One type is a single-copied adult TnI (adTnI) gene, which contains eight exons and seven introns. adTnI expresses two isoforms, the shorter body wall muscle TnI and the longer cardiac TnI, through alternative splicing. The mRNAs of these TnI isoforms may undergo trans-splicing of the 5'-leader sequences, like the TnI mRNA of another ascidian species, Ciona intestinalis. The other type comprises multi-copied larval TnI (laTnI) genes. Halocynthia has at least three laTnIs (alpha, beta, and gamma), which are composed of five exons and four introns, and two of them (alpha and gamma) are clustered in tandem. All laTnIs have B- and M-regions within their 5'-upstream regions, which have been discovered to be the regulatory elements of Halocynthia larval actin genes. The expression of Halocynthia laTnIs and larval actins may be regulated in the same manner. It is known that Ciona does not possess a larva-specific TnI isoform. The phylogenetic tree of ascidian TnIs suggests that laTnIs might have only been generated within the Pleurogona lineage after Enterogona/Pleurogona divergence, and this scenario well agrees with the absence of laTnIs in Ciona.

Tryptophan-catabolizing enzymes - party of three.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that have independently evolved to catalyze the first step in tryptophan catabolism via the kynurenine pathway (KP). The depletion of tryptophan and formation of KP metabolites modulates the activity of the mammalian immune, reproductive, and central nervous systems. IDO and TDO enzymes can have overlapping or distinct functions depending on their expression patterns. The expression of TDO and IDO enzymes in mammals differs not only by tissue/cellular localization but also by their induction by distinct stimuli. To add to the complexity, these genes also have undergone duplications in some organisms leading to multiple isoforms of IDO or TDO. For example, many vertebrates, including all mammals, have acquired two IDO genes via gene duplication, although the IDO1-like gene has been lost in some lower vertebrate lineages. Gene duplications can allow the homologs to diverge and acquire different properties to the original gene. There is evidence for IDO enzymes having differing enzymatic characteristics, signaling properties, and biological functions. This review analyzes the evolutionary convergence of IDO and TDO enzymes as tryptophan-catabolizing enzymes and the divergent evolution of IDO homologs to generate an enzyme family with diverse characteristics not possessed by TDO enzymes, with an emphasis on the immune system.