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

Groom, Katherine; Bhattacharya, Anupam; Zechel, David L.

doi:10.1002/cbic.201000580

Cited by 27 publications

(23 citation statements)

References 25 publications

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“…21–22 It also includes a gene that encodes for a homolog of the cytochrome P450 ( hysP ) that carries out the C β aryl coupling needed to produce the indolocarbazole intermediate ( 4 ) 23 and the flavin-dependent monooxygenase ( hysC ) that directs the formation of the pyrrolinone indolocarbazole core from this intermediate. 24 In addition to these conserved pyrrolinone indolocarbazole biosynthesis genes, the hys cluster contains two genes predicted to encode cytochrome P450 hydroxylases ( hysX1, hysX2 ) that are not seen in any characterized gene clusters. Based on gene content, we predicted that the hys cluster would likely encode for the biosynthesis of a di-hydroxylated pyrrolinone indolocarbazole-based metabolite.…”

Section: Resultsmentioning

confidence: 99%

Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment

et al. 2015

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Natural product discovery from environmental genomes (metagenomics) has largely been limited to the screening of existing environmental DNA (eDNA) libraries. Here, we have coupled a chemical-biogeographic survey of chromopyrrolic acid synthase (CPAS) gene diversity with targeted eDNA library production to more efficiently access rare tryptophan dimer (TD) biosynthetic gene clusters. A combination of traditional and synthetic biology-based heterologous expression efforts using eDNA-derived gene clusters led to the production of hydroxysporine (1) and reductasporine (2), two bioactive TDs. As suggested by our phylogenetic analysis of CPAS genes, identified in our survey of crude eDNA extracts, reductasporine (2) contains an unprecedented TD core structure: a pyrrolinium indolocarbazole core that is likely key to its unusual bioactivity profile. This work demonstrates the potential for the discovery of structurally rare and biologically interesting natural products using targeted metagenomics, where environmental samples are prescreened to identify the most phylogenetically unique gene sequences and molecules associated with these genes are accessed through targeted metagenomic library construction and heterologous expression.

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Section: Resultsmentioning

confidence: 99%

Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment

et al. 2015

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“…The results are reported in an in vivo experiment by Zechel's group. 22) The crystal structure of RebC revealed that Phe-216 and Arg-239 play important roles in substrate binding, via hydrophobic-and hydrogen-bonding interactions respectively. Zechel's and our mutational analyses indicated that these residues are crucial for substrate binding as well as selective arcyriaflavin A formation.…”

Section: Discussionmentioning

confidence: 99%

“…21) However, exogenous FAD causes the acceleration of K252c production, 15) and replacement of F216V and R239N in RebC can confer StaC-type activity on the FAD-dependent monooxygenase RebC. 22) Thus the function of FAD in the production of K252c by StaC remains obscure. In addition, the molecular mechanism of StaC-or RebC-driven single differential product formation remains unclear.…”

mentioning

confidence: 99%

Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis

Asamizu

Shiro

Igarashi

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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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.

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“…[26,52] Groom and co-workers [53] were able to switch the regioselectivity of RebC from rebeccamycin to staurosporine production through exchanging a pair of active-site residues important in indolocarbazole ring contact. Among different indolocarbazole biosynthetic enzymes, Arg239 and Phe216 emerged as the key residues, mediating repeated regioselective substrate oxidation.…”

Section: Flavoprotein Hydroxylase Rebcmentioning

confidence: 98%

Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases

et al. 2011

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Monooxygenases perform chemo-, regioand/or enantioselective oxygenations of organic substrates under mild reaction conditions. These properties and the increasing number of representatives along with effective preparation methods place monooxygenases in the focus of industrial biocatalysis. Mechanistic and structural insights reveal reaction sequences and allow turning them into efficient tools for the production of valuable products. Herein we describe two biocatalytically relevant subclasses of flavoprotein monooxygenases with a close evolutionary relation: subclass A represented by p-hydroxybenzoate hydroxylase (PHBH) and subclass E formed by styrene monooxygenases (SMOs). PHBH family members perform highly regioselective hydroxylations on a wide variety of aromatic compounds. The more recently discovered SMOs catalyze a number of stereoselective epoxidation and sulfoxidation reactions. Mechanistic and structural studies expose distinct characteristics, which provide a promising source for future biocatalyst development.

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

Cited by 27 publications

References 25 publications

Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment

Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment

Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis

Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases

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