Robust
            <i>in vitro</i>
            activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Yeh, Ellen; Garneau, Sylvie; Walsh, Christopher T.

doi:10.1073/pnas.0500755102

Cited by 253 publications

(298 citation statements)

References 51 publications

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“…Recent work within the Walsh group has established that RebF and RebH act as a two-component reductase/ halogenase system (13). RebF is able to generate FADH 2 , and RebH uses this reduced flavin, along with O 2 and chloride ion, to generate an oxidizing halogen equivalent.…”

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confidence: 99%

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

2005

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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...

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

2005

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“…The kinetic parameters for RadH with selected substrates were determined (Table 1 and Figures S4–S10). Notably, the k cat values for RadH with iso‐quinoline 6 and coumarin 8 were significantly higher than those reported for Trp‐Hal enzymes with the natural substrate 3a, 13. Whilst kinetic analysis shows that monocillin II ( 1 ) is turned over more slowly than the other substrates, k cat / K m was not determined for 1 since saturation of the Michaelis–Menten curve was not attained.…”

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confidence: 78%

RadH: A Versatile Halogenase for Integration into Synthetic Pathways

et al. 2017

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Flavin‐dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site‐directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high‐throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin “non‐natural” product in E. coli.

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“…Substituted naphthalenes were also viable substrates, and monochlorinated compounds 10 and 11 were isolated in high yields. While these latter substrates were halogenated at their most activated sites, they nonetheless illustrate the ability of RebH to accept substrates significantly different from tryptophan.To further demonstrate the synthetic utility of RebH, we confirmed that the bromination capabilities of this enzyme [13] could be translated to the preparative scale, and addition of NaBr to the reaction medium provided 7-bromotryptophan (12) in 85% isolated yield. The reaction scale was also increased to enable chlorination of 100 mg of tryptophan using crude cell lysate, rather than purified enzyme, as a catalyst (Scheme 4).…”

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“…Reported values for the k cat and K m of RebH on L-tryptophan are 1.4 min −1 and 2.0 M, [13] respectively.…”

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“…[11] Inspired by these examples, we set out to explore the synthetic utility of RebH in preparative halogenation reactions. We focused on this enzyme and its cognate flavin reductase, RebF, due to reports of their expression in E. coli, [13] which facilitates both enzyme production and genetic manipulation. However, while both RebH and RebF were indeed expressed in E. coli using published procedures, [13,16] low enzyme yields hindered their synthetic utility.…”

Section: Introductionmentioning

confidence: 99%

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Regioselective Arene Halogenation using the FAD‐Dependent Halogenase RebH

2013

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Co-expression of the halogenase RebH with GroEL/ES and fusion of the flavin reductase RebF to MBP enabled production of both enzymes on scales sufficient for preparative regioselective oxidative halogenation of arenes. The activity and selectivity of RebH contrast with those reported for PrnA, a structurally homologous halogenase, which provided a narrower substrate scope and only enabled halogenation of unnatural substrates at their most electronically activated positions. KeywordsRebH; halogenase; regioselective; biocatalysis Halogenated organic compounds are essential building blocks for chemical synthesis [1] and play important roles as pharmaceuticals [2] and agrochemicals [3] . Despite the utility of these compounds, installing halogen atoms frequently necessitates the use of activated or prefunctionalized starting materials and wasteful multistep functional group conversion sequences. [4] Arene halogenation via electrophilic aromatic substitution (EAS) requires harsh reaction conditions using stoichiometric reagents and suffers from poor regioselectivity in many cases. [5] Oxidative methods to catalytically generate halogenating agents from halide salts have been developed, but none of these have solved the aforementioned selectivity problem, and few utilize oxygen as a terminal oxidant (Scheme 1). [6] New, more efficient methods for oxidative halogenation would significantly improve the syntheses of a wide range of chemicals and are therefore highly desirable. [6] Given the high efficiency and selectivity often associated with enzyme-catalyzed reactions, we were drawn to several classes of enzymes that catalyze oxidative halogenation reactions. [7] Particularly notable in this regard are the FAD-dependent halogenases, which were first characterized by van Pée and coworkers in the late 1990's. [8] Extensive mechanistic investigation of these enzymes by both van Pée and Walsh, focusing predominately on tryptophan halogenases, has clarified their mechanism. [9] In short, they utilize FADH 2 supplied by a NAD-dependent Correspondence to: Jared C. Lewis, jaredlewis@uchicago.edu. Supporting information, including full experimental procedures, for this article is available on the WWW under http:// www.angewandte.org or from the author. While FAD-dependent halogenases thus have the potential to improve a number of problems with current oxidative halogenation methods, they remain largely unexplored for preparative synthesis. Characterization of their activity in vitro has typically involved analytical scale reactions of tryptophan, [9] but van Pée and coworkers also explored the substrate scope of the tryptophan-7-halogenase PrnA expressed in Pseudomonas fluorescens (Scheme 2). [11] A variety of substituted indoles were accepted by the enzyme, but halogenation invariably occurred at the electronically most activated indole 2-position for all substrates except tryptophan. These same researchers were able to alter the selectivity of PrnA to produce 5-chloro-and 5-bromotryptophan by mutating a single acti...

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Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Cited by 253 publications

References 51 publications

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

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

RadH: A Versatile Halogenase for Integration into Synthetic Pathways

Regioselective Arene Halogenation using the FAD‐Dependent Halogenase RebH

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