Biochemical and Structural Studies of the Carminomycin 4- O-Methyltransferase DnrK

Abstract

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Figure 1.

A, Overview of the final steps of DNR (1a) and DXR biosynthesis with emphases on the DnrK-catalyzed methylation reaction (box) and DnrK regioselectivity (blue). B, Previously reported DnrK or DauK substrates with MT regioselectivity highlighted (blue).

Figure 2.

A, Representative anthracene, tetracene, benz[a]anthracene, and related DnrK substrates. Highlighted DnrK regioselectivity (blue), where determined, was based on isolation and structure elucidation [4-O-methoxyarancinamycin (7a)] or coelution with product standards (1a, 18, 24, 26, and 32) or assigned based on the presence of a single aromatic hydroxyl (pyranonapthoquinones). B, The percent conversion to product in end point LC-MS assays (1 mM test agent, 3.2 mM AdoMet, 100 μM DnrK, 37 °C, 16 h). Turnover is categorized in the plot and under each structure as a colored ball as good (≥75%, green), moderate (>25% to <75%, orange), low (≤25%, red), and no turnover (gray).

Figure 3.

A, Representative flavones, flavonols, flavanones, isoflavones, coumarins, and related DnrK substrates. Highlighted DnrK regioselectivity (blue), where determined, was based on coelution with product standards (47, 64, 66, 68, 76, 86, and 88) or assigned based on the presence of a single aromatic hydroxyl (45, 46, and 89). B, The percent conversion to product in end-point LC-MS assays (1 mM test agent, 3.2 mM AdoMet, 100 μM DnrK, 37 °C, 16 h). Turnover is categorized in the plot and under each structure as a colored ball as good (≥75%, green), moderate (>25% to <75%, orange), low (≤25%, red), and no turnover (gray). Percent di- and/or trimethylation for compounds 43, 44, and 78 are represented as lighter shades of each represented color.

Figure 4.

A, Representative 2-quinolones, fused benzoic acid containing natural products, and related DnrK substrates. Highlighted DnrK regioselectivity (blue), where determined, was based on the presence of a single aromatic hydroxyl (91, 93, and 94) and detection of monomethylated product or two aromatic hydroxyls (98) and detection of mono- and dimethylated products. B, The percent conversion to product in end-point LC-MS assays (1 mM test agent, 3.2 mM AdoMet, 100 μM DnrK, 37 °C, 16 h). Turnover is categorized in the plot and under each structure as a colored ball as good (≥75%, green), moderate (>25% to >75%, orange), low (≤25%, red), and no turnover (gray). Percent dimethylation for compound 98 is represented as lighter shades of orange.

Figure 5.

A, Dose–response of compounds CAR (1), DNR (1a), aranciamycin (7), 4-O-methoxyaranciamycin (7a), and steffimycin B (9) against A549 (non-small-cell lung) human cancer cell line (72 h). B, Dose–response of mentioned compounds against the PC3 (prostate) human cancer cell line (72 h). A549: IC₅₀ for the compounds (1, 13.09 nM; 1a, 199.0 nM; 7, 2.7 μM; 7a, 13.8 μM; 9, 2.8 μM). PC3: IC₅₀ for the compounds (1, 34.98 nM; 1a, 170.1 nM; 7, 1.1 μM; 7a, 4.3 μM; 9, 3.1 μM). C, HCT116 cells were treated with 2 μM of test compounds or DMSO (negative control) for 6 h followed by Western blot analysis for the indicated proteins. D, The impact of 10 μM steffimycin B (9) on axolotl embryo tail regeneration at 7 dpa compared to vehicle (DMSO, negative control) and the Hsp90 inhibitor geldanamycin (positive control). Compounds 1, 1a, 7, and 7a led to no inhibition of tail regeneration under identical conditions.