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.

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.

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.

Evolution of vertebrate indoleamine 2,3-dioxygenases.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the same reaction, the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed among life-forms, being found not only in eukaryotes but also in bacteria. In contrast, IDO has been found only in mammals and yeast to date. However, recent genome and EST projects have identified IDO homologues in non-mammals and found an IDO paralogue that is expressed in mice. In this study, we cloned the frog and fish IDO homologues and the mouse IDO paralogue, and characterized their enzymatic properties using recombinants. The IDOs of lower vertebrates and the mouse IDO paralogue had IDO activity but had 500-1000 times higher K(m) values and very low enzyme efficiency compared with mammalian IDOs. It appears that L-Trp is not a true substrate for these enzymes in vivo, although their actual function is unknown. On the phylogenetic tree, these low-activity IDOs, which we have named "proto-IDOs," formed a cluster that was distinct from the mammalian IDO cluster. The IDO and proto-IDO genes are present tandemly on the chromosomes of mammals, including the marsupial opossum, whereas only the proto-IDO gene is observed in chicken and fish genomes. These results suggest that (mammalian) IDOs arose from proto-IDOs by gene duplication that occurred before the divergence of marsupial and eutherian (placental) mammals in mammalian evolutionary history.

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.

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.

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.

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.