The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro. In this study, we mutated Ala-118 in StaC by replacing a glutamine that is conserved in FAD monooxygenases, resulting in increased FAD content as well as catalytic activity. In addition, mutations around the substrate-binding sites of StaC and RebC can change the product selectivity. Specifically, StaC-N244R-V246T and RebC-F216V-R239N mutants produced substantial amounts of arcyriaflavin A and K252c, respectively.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1271/bbb.110474 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
An unusual role for a mobile flavin in StaC-like indolocarbazole biosynthetic enzymes.
Chem Biol
July 2012
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
The indolocarbazole biosynthetic enzymes StaC, InkE, RebC, and AtmC mediate the degree of oxidation of chromopyrrolic acid on route to the natural products staurosporine, K252a, rebeccamycin, and AT2433-A1, respectively. Here, we show that StaC and InkE, which mediate a net 4-electron oxidation, bind FAD with a micromolar K(d), whereas RebC and AtmC, which mediate a net 8-electron oxidation, bind FAD with a nanomolar K(d) while displaying the same FAD redox properties. We further create RebC-10x, a RebC protein with ten StaC-like amino acid substitutions outside of previously characterized FAD-binding motifs and the complementary StaC-10x.
View Article and Find Full Text PDFEnzymes involved in natural product biosynthesis employ a variety of cofactors, reaction mechanisms, and substrate preferences to achieve remarkable chemical diversity found in nature. In this issue of Chemistry & Biology, Goldman and colleagues show how cofactor (FAD) binding affinity impacts the reaction mechanism and outcome of two related proteins, RebC and StaC, involved in indolocarbazoles biosynthesis.
View Article and Find Full Text PDFCharacterization and functional modification of StaC and RebC, which are involved in the pyrrole oxidation of indolocarbazole biosynthesis.
Biosci Biotechnol Biochem
March 2012
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Toyama, Japan.
The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro.
View Article and Find Full Text PDFRebeccamycin and staurosporine biosynthesis: insight into the mechanisms of the flavin-dependent monooxygenases RebC and StaC.
Chembiochem
February 2011
Department of Chemistry, Queen's University, 90 Bader Lane, Kingston, Ontario, K7L 3N6, Canada.
Staurosporine and rebeccamycin aglycones are assembled by the oxidative action of StaP, StaC, and RebC on chromopyrrolic acid.
J Am Chem Soc
September 2006
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!