Biosynthesis of Dothideomins Reveals a Fungal P450 That Constructs the Tricyclo[5.2.2.0.4,8]undecane-Imbedded Core Skeleton

Click the “Nanjing IFB” above to follow us!

Hello everyone, today I would like to share a recent article published in theJACS journal titled “Biosynthesis of Dothideomins Reveals a Fungal P450 That Constructs the Tricyclo[5.2.2.0.^4,8]undecane-Imbedded Core Skeleton”, with Professor Li Dehai from the School of Medicine, Ocean University of China as the corresponding author, who is mainly engaged in the research of marine natural product chemistry. This article reveals the biosynthetic process of Dothideomins C and D from the transformation of the anthraquinone emodin, catalyzed by the cytochrome P450 monooxygenase DotG, which forms three C−C bonds, thereby constructing the tricyclo[5.2.2.0.^4,8]undecane core skeleton, followed by the reduction to the final products by the aldehyde ketone reductase DotH and SDR DotM.

Figure 1: The biosynthetic process of Dothideomins C and D

Anthraquinones (AQs), which have a tricyclic core structure (A, B, and C rings), are 9,10-anthraquinones that are biologically derived from the common polyketide precursor emodin, exhibiting a wide range of pharmacological activities such as anti-inflammatory, antibacterial, and hemostatic effects. Dimeric anthraquinones (BQs), produced from the dimerization of anthraquinone monomers, possess even stronger biological activity. Dimeric anthraquinones are formed by the connection of two identical or different anthraquinone monomers via C-C or C-O bonds. Anthraquinone monomers can undergo simple dimerization through one or two cross-links or form complex cage-like structures through three or more cross-links.

Figure 2: Representative dimeric anthraquinones (BQs)

In 2022, the authors isolated four antibacterial cage-like dimeric anthraquinones, Dothideomins A−D, with the embedded tricyclo[5.2.2.0.^4,8]undecane core skeleton formed by the opening of the A ring of emodin and dimerization after the loss of C8. Most AQs undergo ring-opening reactions at the B ring, typically catalyzed by Baeyer−Villiger monooxygenases or dioxygenases, while enzymes responsible for the opening of the A ring have not been reported. Therefore, the authors studied the biosynthetic process of Dothideomins A-D.

First, the authors used P450 rugG as a probe to identify a highly conserved dot gene cluster in the endophytic fungus Dothideomycetes sp. BMC-101 (Figure 4A), and heterologously expressed dotA-H and dotM (Figure 4B, trace (i)) in Aspergillus nidulans (AN), producing rugulosin A, Dothideomins C, and D, thereby determining that the biosynthetic gene cluster for Dothideomins C and D is dot.

Figure 3: Characterization of the synthesis process of Dothideomins C and D

Scheme 1: Predicted biosynthetic process of Dothideomins C and D

Subsequently, to validate the predicted biosynthetic process in Scheme 1, the authors heterologously expressed the genes on the dot gene cluster:

First, expressing dotABEF yielded the main product emodin (1) and 2-4 (Figure 4B, trace (ii)), as dotABEF does not contain oxidases, it is speculated that 2-4 are oxidation products of 1 under the action of endogenous enzymes, thus the authors verified this result through in vitro feeding experiments (Figure 5).

Figure 4: In vitro feeding experiments

Second, to determine whether DotG has the function of catalyzing anthraquinone dimerization, the authors co-expressed dotG with dotABEF (Figure 4B, trace (iii)), isolating 5-8 and 11; similarly, feeding 1 to AN-dotG (Figure 5, trace (iii)) also yielded 5-8 and 11. To determine the relationship of the products 5-8, the authors conducted in vitro feeding experiments (Figure 6B and C), confirming that 6 converts to 7, and 5-7 are degradation products of 8. It was concluded that the final products catalyzed by DotG from 1 are 8 and 11. To determine the active site of DotG, the authors constructed a series of DotG mutants based on sequence alignment and molecular docking results for in vitro feeding experiments (Figure 7), concluding that His114, Trp144, Arg259, Phe311, Asn316, Cys318, Ala321, Trp387, Phe390, Arg394, Pro514 are responsible for substrate structure stability and function, while His119, Asp131, Ile126, Ile120, Val123, His128, Pro130 are involved in substrate binding, among which His119, Asp131, and Ile126 play a role in product selection. The authors also performed density functional theory (DFT) calculations (Figure 8), concluding that the decarboxylation process to form the cage-like structure is the rate-limiting step, followed by rapid cyclization and hydroxyl removal, accompanied by a large amount of energy release.

Figure 5: In vitro feeding experiments of AN-dotG and the relationship between compounds 2-8

Figure 6: In vitro feeding experiments of DotG mutants

Figure 7: DFT calculations

Third, to determine the functions of DotH and DotM, the authors heterologously expressed dotH with dotA-G (Figure 4B, trace (iv)), and conducted in vitro feeding experiments of DotH (Figure 10A), finding that only co-expressing dotH with dotG (Figure 10A, trace (iv)) could yield rugulosin A, Dothideomins C, and D, concluding that DotH utilizes the products of DotG to produce these compounds. To verify this hypothesis, the authors expressed DotH in vitro and conducted enzyme reactions (Figure 9), detecting the production of 9. They also co-expressed dotH with feeding 1, yielding only 11 from Tp_rugG (Figure 10A, trace (ix)), detecting the production of 12. Similarly, to determine the function of DotM, the authors co-expressed dotA-G with dotM (Figure 4B, trace (v)) and conducted in vitro feeding experiments (Figure 10C), concluding that DotM catalyzes the reduction of 9 to 10.

Figure 8: Enzyme reactions of DotH

Figure 9: Functional characterization of DotH and DotM

Finally, the authors conducted antibacterial activity tests on the isolated compounds (Table 1), finding that compounds 9, 10, and 12 exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, while compound 11 showed antibacterial activity only against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

Table 1: Antibacterial activity tests

In summary, the authors discovered the gene cluster responsible for synthesizing Dothideomins C and D in Dothideomycetes sp. BMC-101, elucidating the biosynthetic pathway through heterologous expression, in vivo feeding, and in vitro biochemical experiments, revealing three important enzymes in the conversion of emodin to Dothideomins C and D: P450 DotG, AKR DotH, and SDR DotM. Specifically, P450 DotG is responsible for synthesizing the tricyclo[5.2.2.0.^4,8]undecane core skeleton, involving the opening of the A ring and decarboxylation reactions, followed by two-step reductions facilitated by AKR DotH and SDR DotM to generate C and D. Additionally, three new compounds 6-8 were isolated, with compound 7 featuring a unique 6/6/6/5/6/6 hexacyclic structure. This study not only emphasizes the importance of P450 in the biosynthesis of BQs but also deepens the understanding of how nature produces complex and diverse natural products.

Original link: https://pubs.acs.org/doi/10.1021/jacs.4c18595

Article title: Biosynthesis of Dothideomins Reveals a Fungal P450 That Constructs the Tricyclo[5.2.2.0.^4,8]undecane-Imbedded Core Skeleton

Compiled by: Xu Ke

Long press the image below to recognize the QR code in the image and easily follow us!