Binding of Two Flaviolin Substrate Molecules, Oxidative Coupling, and Crystal Structure of Streptomyces coelicolor A3(2) Cytochrome P450 158A2

Abstract

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 Å) and flaviolin-bound (1.62 Å) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

“…Based on the crystal structure of CYP158A2 complexed with the substrates, Zhao et al (26) proposed that the aryl-aryl coupling proceeds by means of direct hydrogen abstraction or direct bond formation with compound I. Although these mechanisms can explain the reaction by P450 158A2, they are an unlikely for the StaP reaction because, as mentioned, the extensive substrate-StaP interaction limits the substrate dynamics required for direct interaction between the substrate and compound I.…”

“…An aryl-aryl coupling reaction is also reported for P450 158A2 that produces flaviolin dimer (26) and P450 OxyC that is involved in vancomycin biosynthesis (27). Based on the crystal structure of CYP158A2 complexed with the substrates, Zhao et al (26) proposed that the aryl-aryl coupling proceeds by means of direct hydrogen abstraction or direct bond formation with compound I.…”

Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization

“…The involvement of cytochrome P450s in the dimerization of napthoquinones, coumarins, and diketopiperazine alkaloid was reported in bacteria, Aspergillus niger, and Aspergillus flavus, respectively (29)(30)(31). In S. coelicolor A3(2), flaviolin is dimerized by a cytochrome P450 (30), and a similar enzyme in Streptomyces griseus dimerizes tetrahydroxynapthalene into 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (32).…”

“…In S. coelicolor A3(2), flaviolin is dimerized by a cytochrome P450 (30), and a similar enzyme in Streptomyces griseus dimerizes tetrahydroxynapthalene into 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (32). In A. niger, the cytochrome P450 KtnC dimerizes dimethyl siderin into kotanin (29).…”

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

“…As mentioned above, many pathways of bacterial and fungal secondary metabolism contain CYPs involved in multi-step oxidation, rearrangements, epoxidations and heteroatom oxidation. Microbial CYPs can contribute other activities in secondary metabolism, such as carbon-carbon bond formation in flaviolin polymer biosynthesis involving CYP158 [133]. These diverse reactions are important for the activity of the final compounds and their diversity (table 1).…”