Enzymatic Assembly of the Bis-Indole Core of Rebeccamycin

Abstract

Rebeccamycin is a member of the family of indolocarbazole antibiotics with broad spectrum antitumor activity. The indolocarbazole framework is derived from two molecules of tryptophan, but very little is known about the enzymes involved in rebeccamycin biosynthesis. Here, we show that RebD is responsible for all catalytic steps forming the central pyrrole ring of chlorochromopyrrolic acid from two molecules of chloroindolepyruvic acid. This transformation does not require any additional cofactors and constitutes the first step of bis-indole formation in the biosynthesis of rebeccamycin.

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

“…Biochemical characterizations of the biosyntheses of staurosporine or rebeccamycin have been reported for several enzymes (7)(8)(9)(10). However, until now, the only crystal structure available was that of RebH which provides 7-chlorotryptophan, a precursor of rebeccamycin (11).…”

Rebeccamycin and Staurosporine Biosynthesis: Insight into the Mechanisms of the Flavin‐Dependent Monooxygenases RebC and StaC

“…[1][2][3] Two archetypal representatives of this class, rebeccamycin (1) and staurosporine (2), exemplify the astonishing range of mechanistically distinct bioactivities that can be achieved with the indolocarbazole scaffold, the former as a DNA topoisomerase I poison, the latter as a protein kinase inhibitor. Identification of the bacterial biosynthetic genes for 1 [4] and 2, [5] along with in vitro studies on individual enzymes' activities, [6][7][8][9] has delineated a four-enzyme pathway to assemble the corresponding indolocarbazoles arcyriaflavin A (6) and K252c (7). As summarized in Scheme 1, these enzymes are RebODPC and StaODPC for 6 and 7, respectively.…”

“…RebD, a heme-dependent oxidase, subsequently oxidatively couples two equivalents of the imine form of indole-3-pyruvate to form chromopyrrolic acid (3). [6,7,9] Compound 3 is moderately stable and slowly oxidizes to form 6, 7, and 7-hydroxyK252c (8) in low yields. [8] A cytochrome P450 enzyme, RebP, is thought to accelerate this spontaneous reaction by oxidatively coupling the 5 and 5' indole carbons of 3 to generate a labile intermediate 4, [10,11] which can likewise undergo spontaneous oxidative decarboxylation to form 6-8 in greater yields, with 8 being the major product.…”

Crystallography gets the jump on the enzymologists

“…Tryptophan is used as the starting compound for many indolocarbazoles (2,3). Recently, substantial progress has been made in understanding the detailed pathways for the biosynthesis of rebeccamycin and its close relative staurosporine (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Rebeccamycin is an antitumor agent that binds to DNAtopoisomerase I complexes to prevent replication of DNA (16).…”

“…1A). The overall conversion of tryptophan to rebeccamycin consists of several unique biochemical transformations; it involves RebH and RebF, a two-component flavin-dependent enzyme system that halogenates tryptophan (10,11), a flavoenzyme amino acid oxidase, RebO (12), a cytochrome P450 (RebD) that catalyzes the oxidative initial linking of two tryptophan skeletons (6,13,14), and a second cytochrome P450, RebP, that participates with a putative flavoprotein, RebC, to form the basic aglycone scaffold (Fig. 1 A) (7,15).…”