A new regime of heme-dependent aromatic oxygenase superfamily.

Two histidine-ligated heme-dependent monooxygenase proteins, TyrH and SfmD, have recently been found to resemble enzymes from the dioxygenase superfamily currently named after tryptophan 2,3-dioxygenase (TDO), that is, the TDO superfamily. These latest findings prompted us to revisit the structure and function of the superfamily. The enzymes in this superfamily share a similar core architecture and a histidine-ligated heme. Their primary functions are to promote O-atom transfer to an aromatic metabolite. TDO and indoleamine 2,3-dioxygenase (IDO), the founding members, promote dioxygenation through a two-step monooxygenation pathway. However, the new members of the superfamily, including PrnB, SfmD, TyrH, and MarE, expand its boundaries and mediate monooxygenation on a broader set of aromatic substrates. We found that the enlarged superfamily contains eight clades of proteins. Overall, this protein group is a more sizeable, structure-based, histidine-ligated heme-dependent, and functionally diverse superfamily for aromatics oxidation. The concept of TDO superfamily or heme-dependent dioxygenase superfamily is no longer appropriate for defining this growing superfamily. Hence, there is a pressing need to redefine it as a heme-dependent aromatic oxygenase (HDAO) superfamily. The revised concept puts HDAO in the context of thiol-ligated heme-based enzymes alongside cytochrome P450 and peroxygenase. It will update what we understand about the choice of heme axial ligand. Hemoproteins may not be as stringent about the type of axial ligand for oxygenation, although thiolate-ligated hemes (P450s and peroxygenases) more frequently catalyze oxygenation reactions. Histidine-ligated hemes found in HDAO enzymes can likewise mediate oxygenation when confronted with a proper substrate.

In vitro characterization of echinomycin biosynthesis: formation and hydroxylation of L-tryptophanyl-S-enzyme and oxidation of (2S,3S) ß-hydroxytryptophan.

Quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) feature in quinomycin family and confer anticancer activity. In light of the significant potency against cancer, the biosynthetic gene clusters have been reported from many different Streptomyces strains, and the biosynthetic pathway were proposed mainly based on the in vivo feeding experiment with isotope labeled putative intermediates. Herein we report another gene cluster from Streptomyces griseovariabilis subsp. bandungensis subsp. nov responsible for the biosynthesis of echinomycin (a member of quinomycin family, also named quinomycin A) and presented in vitro evidence to corroborate the previous hypothesis on QXC biosynthesis, showing that only with the assistance of a MbtH-like protein Qui5, did the didomain NRPS protein (Qui18) perform the loading of a L-tryptophan onto its own PCP domain. Particularly, it was found that Qui5 and Qui18 subunits form a functional tetramer through size exclusion chromatography. The subsequent hydroxylation on ß-carbon of the loaded L-tryptophan proved in vitro to be completed by cytochrome P450-dependent hydroxylase Qui15. Importantly, only the Qui18 loaded L-tryptophan can be hydroxylated by Qui15 and the enzyme was inactive on free L-tryptophan. Additionally, the chemically synthesized (2S,3S) ß-hydroxytryptophan was detected to be converted by the tryptophan 2,3-dioxygenase Qui17 through LC-MS, which enriched our previous knowledge that tryptophan 2,3-dioxygenase nearly exclusively acted on L-tryptophan and 6-fluoro-tryptophan.

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.