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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 k cat values of metazoan TILs were low, < 1/200 of the k cat of Escherichia coli TIL. By contrast, metazoan TILs showed lower K m 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 K m 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. [Display omitted] • A few metazoan TILs have a spliceosomal intron(s). • Metazoan TILs show weak but obvious activity. • K m -values of metazoan TILs are smaller than those of common bacterial TILs. • High L-Trp affinity may have been acquired by metazoan TILs during evolution. [ABSTRACT FROM AUTHOR]

Abstract: 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 α- and β-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. His74, His288, and His332 were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His332 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. His74 and His288 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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]