Divergence of Classical and C-Ring-Cleaved Angucyclines: Elucidation of Early Tailoring Steps in Lugdunomycin and Thioangucycline Biosynthesis

Abstract

Angucyclines are an important group of microbial natural products that display tremendous chemical diversity. Classical angucyclines are composed of a tetracyclic benz[a]anthracene scaffold with one ring attached at an angular orientation. However, in atypical angucyclines, the polyaromatic aglycone is cleaved at A-, B-, or C-rings, leading to structural rearrangements and enabling further chemical variety. Here, we have elucidated the branching points in angucycline biosynthesis leading toward cleavage of the C-ring in lugdunomycin and thioangucycline biosynthesis. We showed that 12-hydroxylation and 6-ketoreduction of UWM6 are shared steps in classical and C-ring-cleaved angucycline pathways, although the bifunctional 6-ketoreductase LugOIIred harbors additional unique 1-ketoreductase activity. We identified formation of the key intermediate 8-O-methyltetrangomycin by the LugN methyltransferase as the branching point toward C-ring-cleaved angucyclines. The final common step in lugdunomycin and thioangucycline biosynthesis is quinone reduction, catalyzed by the 7-ketoreductases LugG and TacO, respectively. In turn, the committing step toward thioangucyclines is 12-ketoreduction catalyzed by TacA, for which no orthologous protein exists on the lugdunomycin pathway. Our results confirm that quinone reductions are early tailoring steps and, therefore, may be mechanistically important for subsequent C-ring cleavage. Finally, many of the tailoring enzymes harbored broad substrate promiscuity, which we utilized in combinatorial enzymatic syntheses to generate the angucyclines SM 196 A and hydranthomycin. We propose that enzyme promiscuity and the competition of many of the enzymes for the same substrates lead to a branching biosynthetic network and formation of numerous shunt products typical for angucyclines rather than a canonical linear metabolic pathway.

Figure 1

Diversification of angucycline antibiotics. (A) Reaction scheme leading to the formation of the products from classical angucycline pathways landomycin A (1) and gaudimycin C (2), A-ring-cleaved gaudimycin D (3), B-ring-cleaved jadomycin (4), C-ring-cleaved lugdunomycin (5), oleaceran/elmonin (6), and dimerized thioangucycline TAC-A (11). (B) Comparison of selected angucycline-producing biosynthetic gene clusters. Legend: lug, lugdunomycin; tac, thioangucycline; pga, gaudimycin; jad, jadomycin; lan, landomycin. (C) A phylogenetic tree of selected SDR-family enzymes from angucycline BGCs. Investigated 1-, 6-, 7-, and 12-ketoreductases are highlighted in blue, green, red, and magenta, respectively.

Figure 2

Analyses of 1- and 6-ketoreduction activities of LugOIIred and LanV. (A) Comparative analysis of 6-ketoreduction activity in a coupled assay with PgaE using 8 as a substrate demonstrates that both LugOIIred and LanV catalyze the formation of 9. This is in contrast to a reaction with PgaE and UrdMred that converts 8 to 2. (B) Investigation of the substrate promiscuity for 1-ketoreduction using diverse angucyclinone substrates 12–15. LugOIIred converted all substrates to corresponding products 16–19, while no 1-ketoreduction activity was detected for LanV. All HPLC chromatogram traces were recorded at 256 nm.

Figure 3

Analysis of the 8-O-methylation activity of LugN. Conversion of the substrate 7 into product 13 sequentially by the 12-hydroxylase PgaE and the 8-O-methyltransferase LugN. Addition of the 6-ketoreductases LanV or LugOII together with PgaE and LugN directs the transformation of the substrate 7 to 12. However, the additional 1-ketoreduction activity of LugOII results in the accumulation of 16 as the main product. The HPLC chromatograms were recorded at 256 nm. The structures of 12 and 13 were verified using authentic standards, and compounds 14 and 15 were verified by NMR.

Figure 4

Analysis of the 7-ketoreduction activity of LugG and TacO. LugG does not have enzymatic activity on 7, but a coupled reaction with PgaE, LanV, LugN, and LugG convert 7 into the product 20. LugG and TacO are orthologous and convert 12 to 20. The HPLC chromatogram traces were recorded at 256 nm.

Figure 5

Analysis of the 12-ketoreduction activity of TacA. The substrate 7 is converted to a mixture of products 24, 25, and 26 by PgaE, LanV, LugN, and TacA. The 12-ketoreductase TacA harbors broad substrate specificity and can transform various angucyclinone intermediates 12, 13, 14, 15, 21, 22, and 10 to products 24, 25, 26, 27, 28, 29, and 23, respectively. The structure of 24 was elucidated by NMR spectroscopy, while the structures of 25, 26, 27, 28, 29, and 23 were deduced based on the changes in retention time and UV–vis spectra (Figure S41) indicating quinone reduction. The HPLC chromatogram traces were recorded at 256 nm.

Figure 6

Combinatorial enzymatic reactions for the production of hydranthomycin (30) and SM 196 A (31). Substrate 12 was converted into 30 and 31 in two-step reactions (labeled with 1. and 2. in the chromatogram traces) with different combinations of ketoreductases LugOIIred, LugG, TacA, and TacO. The HPLC chromatogram traces were recorded at 256 nm.