Publications

• Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

  Onaka H., Taniguchi S., Igarashi Y., Furumai T.

  The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examine ... >> More

  The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes. << Less

  Biosci. Biotechnol. Biochem. 67:127-138(2003) [PubMed] [EuropePMC]

• Enzymatic generation of the chromopyrrolic acid scaffold of rebeccamycin by the tandem action of RebO and RebD.

  Howard-Jones A.R., Walsh C.T.

  During the biosynthesis of the fused six-ring indolocarbazole scaffolds of rebeccamycin and staurosporine, two molecules of L-tryptophan are processed to a pyrrole-containing five-ring intermediate known as chromopyrrolic acid. We report here the heterologous expression of RebO and RebD from the r ... >> More

  During the biosynthesis of the fused six-ring indolocarbazole scaffolds of rebeccamycin and staurosporine, two molecules of L-tryptophan are processed to a pyrrole-containing five-ring intermediate known as chromopyrrolic acid. We report here the heterologous expression of RebO and RebD from the rebeccamycin biosynthetic pathway in Escherichia coli, and tandem action of these two enzymes to construct the dicarboxypyrrole ring of chromopyrrolic acid. Chromopyrrolic acid is oxidized by six electrons compared to the starting pair of L-tryptophan molecules. RebO is an L-tryptophan oxidase flavoprotein and RebD a heme protein dimer with both catalase and chromopyrrolic acid synthase activity. Both enzymes require dioxygen as a cosubstrate. RebD on its own is incompetent with L-tryptophan but will convert the imine of indole-3-pyruvate to chromopyrrolic acid. It displays a substrate preference for two molecules of indole-3-pyruvic acid imine, necessitating a net two-electron oxidation to give chromopyrrolic acid. << Less

  Biochemistry 44:15652-15663(2005) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Molecular analysis of the rebeccamycin L-amino acid oxidase from Lechevalieria aerocolonigenes ATCC 39243.

  Nishizawa T., Aldrich C.C., Sherman D.H.

  Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in el ... >> More

  Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by rebO, located at the left-hand region of the reb gene cluster. The deduced protein product, RebO (51.9 kDa), is an L-amino acid oxidase (L-AAO) that has 27% identity to an L-AAO from Scomber japonicus (animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the rebO gene was overexpressed and purified from Escherichia coli. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-L-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products. << Less

  J. Bacteriol. 187:2084-2092(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.