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.