Staurosporine and Rebeccamycin Aglycones Are Assembled by the Oxidative Action of StaP, StaC, and RebC on Chromopyrrolic Acid

J Am Chem Soc. 2006 Sep 20;128(37):12289-98. doi: 10.1021/ja063898m.

Abstract

In the biosynthesis of the antitumor indolocarbazoles rebeccamycin and staurosporine by streptomycetes, assembly of the aglycones involves a complex set of oxidative condensations. Overall formation of aglycones K252c and arcyriaflavin A from their biosynthetic precursor chromopyrrolic acid involves four- and eight-electron oxidations, respectively. This process is catalyzed by the remarkable enzyme StaP, with StaC and RebC acting to direct the level of oxidation in the newly formed five-membered ring. An aryl-aryl coupling reaction is integral to this transformation as well as oxidative decarboxylation of the dicarboxypyrrole moiety of chromopyrrolic acid. Herein we describe the heterologous expression of staP, staC, and rebC in Escherichia coli and the activity of the corresponding enzymes in constructing the two distinct six-ring scaffolds. StaP is a cytochrome P450 enzyme, requiring dioxygen, ferredoxin, flavodoxin NADP(+)-reductase, and NAD(P)H for activity. StaP on its own converts chromopyrrolic acid into three aglycone products, K252c, arcyriaflavin A, and 7-hydroxy-K252c; in the presence of StaC, K252c is the predominant product, while the presence of RebC directs formation of arcyriaflavin A. (18)O-Labeling studies indicate that the oxygen(s) of the pyrrolinone and maleimide functionalities of the aglycones formed are all derived from dioxygen. This work allowed for the in vitro reconstitution of the full biosynthetic pathway from l-tryptophan to the staurosporine and rebeccamycin aglycones, K252c and 1,11-dichloroarcyriaflavin A.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Carbazoles / metabolism*
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / isolation & purification
  • Cytochrome P-450 Enzyme System / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Flavin-Adenine Dinucleotide / chemistry
  • Flavin-Adenine Dinucleotide / metabolism
  • Indoles / metabolism*
  • Kinetics
  • Mass Spectrometry
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / isolation & purification
  • Mixed Function Oxygenases / metabolism
  • NAD / chemistry
  • NAD / metabolism
  • Oxidation-Reduction
  • Oxygen Isotopes
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Staurosporine / biosynthesis*
  • Streptomycetaceae / enzymology
  • Streptomycetaceae / genetics
  • Streptomycetaceae / metabolism

Substances

  • Carbazoles
  • Indoles
  • Oxygen Isotopes
  • Recombinant Proteins
  • NAD
  • Flavin-Adenine Dinucleotide
  • Cytochrome P-450 Enzyme System
  • rebeccamycin
  • Mixed Function Oxygenases
  • Staurosporine