C-N-Coupled Metabolites Yield Insights into Dynemicin A Biosynthesis

Abstract

The biosynthesis of the three structural subclasses of enediyne antitumor antibiotics remains largely unknown beyond a common C₁₆ -hexaene precursor. For the anthraquinone-fused subtype, however, an unexpected iodoanthracene γ-thiolactone was established to be a mid-pathway intermediate to dynemicin A. Having deleted a putative flavin-dependent oxidoreductase from the dynemicin biosynthetic gene cluster, we can now report four metabolites that incorporate the iodoanthracene and reveal the formation of the C-N bond linking the anthraquinone and enediyne halves emblematic of this structural subclass. The coupling of an aryl iodide and an amine is familiar from organometallic chemistry, but has little or no precedent in natural product biosynthesis. These metabolites suggest further that enediyne formation occurs early in the overall biosynthesis, and that even earlier events might convert the C₁₆ -hexaene to a common C₁₅ intermediate that partitions to enediyne and anthraquinone building blocks for the heterodimerization.

Keywords: biosynthesis; dynemicin; enediyne; oxidoreductases; polyketides.

Figure 1.

Representative structural types of enediyne antitumor antibiotics: Neocarzinostatin (NCS, 1), calicheamicin (CLM, 2), and dynemicin A (DYN, 3). The dashed line in 3 represents the junction of the two halves of DYN, each of separate polyketide origin.

Figure 2.

Biogenetic proposal for coupling the enediyne and anthraquinone precursors of DYN. DynE8 assembles β-hydroxyhexaene 4, which is processed by the enediyne TE to generate heptaene 5. The C₁₆-hexaene 4 is also believed to be an intermediate en route to both the enediyne and anthraquinone halves of DYN (X and iodoanthracene 6, respectively), which are coupled to generate the heterodimeric natural product.

Figure 3.

a) HPLC comparison of fermentation extracts from ΔE13 and wild-type M. chersina, and a ΔE13 reconstitution strain expressing C-terminally hexahistidine-tagged E13. b) UV-vis spectra, solution images, masses, and predicted molecular formulae of 7-10

Figure 4.

Rationale for the production of 7–10 by M. chersina ΔE13. a) β-hydroxyhexaene 4 is proposed to partition to enediyne precursor X and iodoanthracene 6 (through a possibly common C_l5-intermediate Z), which are coupled to generate the on-pathway anthracene-enediyne Y. b) In the presence of E13, Y is processed to DYN; in its absence, this mutant would be expected to accumulate 13, its possible substrate, which would rapidly isomerize to 10. Carbons of 13 indicated with a dot (•) would be lost en route to DYN. c) Alternatively, epoxidation could occur just after the double bond is formed at the D/F-ring fusion in an enediyne precursor, and in its absence 10 could be formed directly in the heterodimerization event.

Figure 5.

Structure of putative anthraquinone-enediyne 11. Reincorporation of shunt metabolite 10 by wild-type M. chersina yielded several minor products, including compound 11, whose proposed structure is based on exact mass determination and UV-vis comparison to dideoxydynemicin A (12).