Enzymatic Generation of the Chromopyrrolic Acid Scaffold of Rebeccamycin by the Tandem Action of RebO and RebD

Abstract

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.Rebeccamycin 1 and staurosporine 2 ( Figure 1) are indolocarbazole antibiotics originally isolated from the actinomycetes LecheValieria aerocolonigenes and Streptomyces longisporoflaVus, respectively. Rebeccamycin is an inhibitor of DNA topoisomerase I, with a minimum inhibitory concentration of 1.75 µM (1). Staurosporine is one of the strongest inhibitors of protein kinases, displaying an IC 50 of 2.7 nM for protein kinase C (2) and IC 50 's in the range of 1-20 nM for most protein kinases. Because of the importance of both the protein kinases and DNA topoisomerases in cell growth and proliferation, these two compounds have been extensively studied as antitumor drug candidates. Analogues of both rebeccamycin and staurosporine have entered clinical trials for the treatment of neoplastic tumors (3, 4), renal cell cancer (5), and leukemia (6).The fused six-ring indolocarbazole scaffold in 1 and 2 is representative of a variety of natural products and derived from the dimerization of two molecules of L-tryptophan (LTrp) 1 via a complex set of oxidative transformations (7-10) (Scheme 1). Since this natural product scaffold is featured in molecules with an interesting range of biological activities, there has been substantial interest in understanding the enzymology of the oxidative dimerization and ring fusion from the starting pair of L-Trp substrates. Subsequent N-glycosylation of the aglycone through the anomeric carbon of a glucose moiety (in the case of rebeccamycin) and through both C 1 and C 5 of L-ristosamine, the deoxyhexose sugar of staurosporine, followed by the action of methyltransferases (11), leads to the active natural products (Scheme 1). In the case of staurosporine, the enzymatic generation of the N-C linkage between one indole nitrogen and C5 of the aminodeoxyhexose is of notable interest as a novel transformation (11,12).The biosynthetic gene clusters for rebeccamycin and staurosporine have been sequenced and functionally expressed in the heterologous host Escherichia coli, validatin...

Structure and Mechanism of the Rebeccamycin Sugar 4′-O-Methyltransferase RebM

“…1B). As exemplified by the rebeccamycin core reactions these include RebH-catalyzed tryptophan halogenation (12), RebO/RebD-mediated chromopyrrolic acid formation (12)(13)(14), and RebC/RebP-catalyzed ring closure (5,15,16). Similar studies have delineated the catalysts responsible for the key structural features that mechanistically distinguish the indolocarbazoles including the enzymes to form and attach novel sugars (11,17,18) and a culminating series of S-adenosylmethionine (AdoMet) 3 -dependent N-and O-methylations (11,17,18).…”

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

“…1) (7)(8)(9). Subsequently, oxidative coupling of two imines by VioB, RebD, or StaD results in the formation of a short-lived compound that was proposed to be an IPA imine dimer (7,10). For the synthesis of rebeccamycin and staurosporine, this reactive intermediate is spontaneously converted into chromopyrrolic acid (11)(12)(13).…”

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).…”