An Enzymatic Oxidation Cascade Converts δ-Thiolactone Anthracene to Anthraquinone in the Biosynthesis of Anthraquinone-Fused Enediynes

Abstract

Anthraquinone-fused enediynes are anticancer natural products featuring a DNA-intercalating anthraquinone moiety. Despite recent insights into anthraquinone-fused enediyne (AQE) biosynthesis, the enzymatic steps involved in anthraquinone biogenesis remain to be elucidated. Through a combination of in vitro and in vivo studies, we demonstrated that a two-enzyme system, composed of a flavin adenine dinucleotide (FAD)-dependent monooxygenase (DynE13) and a cofactor-free enzyme (DynA1), catalyzes the final steps of anthraquinone formation by converting δ-thiolactone anthracene to hydroxyanthraquinone. We showed that the three oxygen atoms in the hydroxyanthraquinone originate from molecular oxygen (O₂), with the sulfur atom eliminated as H₂S. We further identified the key catalytic residues of DynE13 and A1 by structural and site-directed mutagenesis studies. Our data support a catalytic mechanism wherein DynE13 installs two oxygen atoms with concurrent desulfurization and decarboxylation, whereas DynA1 acts as a cofactor-free monooxygenase, installing the final oxygen atom in the hydroxyanthraquinone. These findings establish the indispensable roles of DynE13 and DynA1 in AQE biosynthesis and unveil novel enzymatic strategies for anthraquinone formation.

Figure 1

AQE biosynthetic gene clusters (BGCs) and the putative roles of DynE13 and DynA1 in AQE biosynthesis. (a) BGCs for sungeidine (sgd) and dynemicin (dyn) biosynthesis. sgd BGC lacks several genes (shown in violet) that are conserved in dyn and other canonical AQE BGCs. The warhead cassette genes essential for enediyne biosynthesis are shown in yellow. (b) The putative function of DynE13 and A1 is based on previous in vivo gene knockout and pathway retrofitting studies.^,,

Figure 2

In vitro enzymatic assays for DynE13 and DynA1. (a,b) The HPLC analysis of the enzymatic conversion of 5 to 6 was performed using the cell-free system (a) and purified enzymes (b). (c) HPLC analysis of the enzymatic activity of DynE13 and DynA1 using 7 as the substrate using the cell-free system. (d) HPLC analysis of the enzymatic conversion of 8 to 9 using the cell-free system. Note that the samples in panels (a) or (c) and (b) or (d) were analyzed by using different HPLC elution conditions. The UV–vis detector of the HPLC was set at 284 nm. (e) Structures of the enzymatic substrates and products.

Figure 3

HPLC analysis of in vivo conversion of 5 in Micromonospora sp. MD118A mutant strains. The mutant strains were fermented in an M5 culture medium without adding the iodide salt that is essential for Sgd and Dyn production. The HPLC detector was set to 550 nm.

Figure 4

Experiments to determine the origin of the oxygen atoms in the hydroxyanthraquinone moiety and the fate of the sulfur in δ-thiolactone anthracene. (a) LC–MS analysis of product 6 in ¹⁸O₂- and H₂¹⁸O-labeling experiments. The ¹⁸O₂-labeling experiment was performed in a cell-free system, while the H₂¹⁸O experiment was first conducted in the cell-free system (approximate H₂¹⁸O/H₂O ratio 1:1) and then using purified enzymes (H₂¹⁸O/H₂O ratio >9:1). (b) Detection of the byproduct H₂S in the DynE13-catalyzed reaction (at a 1 h time point) using the monobromobimane (MBB)-based derivatization method. (c) Detection of H₂S production in the DynE13-catalyzed reaction with the mass spectrum of thiol-DMM adduct shown. The detection of H₂S was performed using an enzyme-coupled method (Scheme S8). (d) Origin of oxygen atoms and the fate of the sulfur group in the anthraquinone biosynthesis from 5 to 6.

Figure 5

Identification of the catalytic residues of DynE13 and DynA1. (a) Model of the DynE13–substrate complex showing the four potential catalytic residues positioned in the proximity of substrate 5. (b) Relative enzymatic activity of DynE13 mutants (T46A, Y179F, C297A, and Y216F). Substrate conversion was monitored by HPLC (Scheme S9). (c) Crystal structure of apo-DynA1 (gray) superimposed on the DynA1 model (pale green) generated by AlphaFold2. The 19-residue motif missing in the crystal structure is shown in yellow. The putative substrate-binding pocket is shown as an orange blob. (d,e) Computational docking generated a model of the DynA1–product complex with the two potential catalytic residues highlighted. The O₂ and H₂O molecules were modeled manually with the molecules connected by a hydrogen-bonding network. (f) HPLC analysis of the enzymatic activity of DynA1 and the two single mutants.

Figure 6

Hypothetical mechanism for the DynE13- and DynA1-catalyzed transformation of δ-thiolactone anthracene into hydroxyanthraquinone. The oxygenation of δ-thiolactone anthracene at C5 and C3 is catalyzed by the flavo-oxygenase DynE13. The oxygenation at C10 is catalyzed by the cofactor-free oxygenase DynA1.