flavin-adenine-dinucleotide and rebeccamycin

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.

Topics: Amino Acid Sequence; Binding Sites; Carbazoles; Cloning, Molecular; Flavin-Adenine Dinucleotide; Indole Alkaloids; Indoles; Molecular Sequence Data; Molecular Structure; Oxidation-Reduction; Oxygenases; Pyrroles; Staurosporine; Streptomyces; Substrate Specificity

The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH(2) (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH(2) and O(2) in the halogenase active site was presumed to form the typical FAD(C4a)-OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)-OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)-OH intermediate. The order of addition of FADH(2) and O(2) was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Formation of flavin intermediates did not require tryptophan, nor were their rates of formation affected by the presence of tryptophan, suggesting that tryptophan likely does not react directly with any flavin intermediates. Furthermore, although final oxidation to FAD occurred with a rate constant of 0.12 s(-)(1), quenched-flow kinetic data showed that the rate constant for 7-chlorotryptophan formation was 0.05 s(-)(1) at 25 degrees C. The kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions. These findings are consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH(2), chloride ion, and O(2).

Topics: Actinomycetales; Carbazoles; Flavin-Adenine Dinucleotide; Flavins; Indoles; Kinetics; Oxidation-Reduction; Oxidoreductases; Tryptophan

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.

Topics: Carbazoles; Cloning, Molecular; Cytochrome P-450 Enzyme System; Escherichia coli; Flavin-Adenine Dinucleotide; Indoles; Kinetics; Mass Spectrometry; Mixed Function Oxygenases; NAD; Oxidation-Reduction; Oxygen Isotopes; Recombinant Proteins; Staurosporine; Streptomycetaceae

The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a k(cat) of 1.4 min(-1) was observed for the RebF/RebH system but increased at least 10-fold in low-pO2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.

Topics: Carbazoles; Chromatography, High Pressure Liquid; Flavin-Adenine Dinucleotide; Indoles; Kinetics; Oxidoreductases; Tryptophan