Examining Heterodimerization by Aryl C–N Coupling in Dynemicin Biosynthesis

Abstract

Distinct among the enediyne antitumor antibiotics, the dynemicin subgroup is comprised of two discrete halves, an enediyne and an anthraquinone, but each is ultimately derived from the same linear β-hydroxyhexaene precursor. The linkage of these two halves by an aryl C–N bond is examined here using a variety of experimental approaches. We demonstrate that this heterodimerization is specific for anthracenyl iodide as the corresponding bromo- and amino-substituted anthracenes do not support dynemicin biosynthesis. Furthermore, biochemical experiments and chemical model reactions support an SRN1 mechanism for the aryl C–N coupling in which electron transfer occurs to the iodoanthracene, followed by loss of an anthracenyl iodide and partition of the resulting aryl radical between C–N coupling and reduction by hydrogen abstraction. An enzyme pull-down experiment aiming to capture the protein(s) involved in the coupling reaction is described in which two proteins, Orf14 and Orf16, encoded by the dynemicin biosynthetic gene cluster, are specifically isolated. Deletion of orf14 from the genome abolished dynemicin production accompanied by a 3-fold increased accumulation of the iodoanthracene coupling partner, indicating the plausible involvement of this protein in the heterodimerization process. On the other hand, the deletion of orf16 only reduced dynemicin production to 55%, implying a noncatalytic, auxiliary role of the protein. Structural comparisons using AlphaFold imply key similarities between Orf14 and X-ray crystal structures of several proteins from enediyne BGCs believed to bind hydrophobic polyene or enediyne motifs suggest Orf14 templates aryl C–N bond formation during the central heterodimerization in dynemicin biosynthesis.

Normal and Aberrant Methyltransferase Activities Give Insights into the Final Steps of Dynemicin A Biosynthesis

“…The appearance of an amine in 22 might be thought to represent the unknown aryl C–N bond formation that initiates the heterodimerization. Recent experiments with synthetic aminoanthracene 23 , however, firmly established that it is not involved in DYN biosynthesis, and indeed, studies reported in Ma et al suggest that 22 is likely a degradation product arising from pathway intermediates or shunt products . In the development of a unified mechanistic understanding of AFE biosynthesis and of DYN ( 1 ) in particular, structure 21 is most germane because when dynA2 was co-expressed in MD118AΔΔ:: dynE13 / A1 / A2 , small amounts of 21 and 22 were again observed but dominated by the production of 24 (Scheme ), in which the pivotal C8–C9 bond had formed to accomplish the second of the heterodimerization reactions that link the “upper” and “lower” halves of the AFEs.…”

“…The Beginning with the DynE8 HR-PKS product 6 and heptaene 7 (Figure 1b), the pathway bifurcates at C 15 to the heterodimerization of iodoanthracene 8 with an aminoenediyne intermediate 9, whose structure is not fully known. 9 A mechanism is proposed to complete linking the "upper" and "lower" halves of AFEs from iodoanthracene−γ−thiolactone 8 and a rationale for the last steps of DYN biosynthesis. 36 to directly generate the centrally important epoxide 37, whose formation would otherwise be exceedingly difficult to rationalize.…”

“…15 On the other hand, iodoanthracene 8 (Figure 1b), bearing a fused γ-thiolactone, is the precursor of the anthraquinone "half" of DYN 16 in steps that ultimately result in all the oxygens in rings A−D being introduced from molecular oxygen, 11 but otherwise the biosynthesis, like its enediyne partner 9, is wholly unknown. 9 Bioinformatic analysis of the DYN BGC revealed not one apparent S-adenosyl-L-methionine (SAM)-dependent methyltransferase but two, DynO6 and DynA5. DynO6 shows homology to tiancimycin TnmH 12 and calicheamicin CalO6, 17 whose different substrate preferences are well described, as well as yangpumicin YpmJ2, suggesting that DynO6 could prove to be responsible for the introduction of the F-ring methoxy in DYN biosynthesis.…”

“…That is, the joining of the "upper" enediyne half and the "lower" anthracene/anthraquinone half involved aryl C−N bond formation. 9 A subsequent process would link C8 and C9 to complete the heterodimerization and lead to DYN and the other known AFEs.…”

Hot off the Press

“…The heterodimerisation step leading to the highly cytotoxic enediyne natural product dynemicin A 21 has been studied, and shown to involve an anthracenyl iodide and a radical nucleophilic substitution mechanism. 21 Enzyme pull-down experiments identified two proteins, Orf14, which is central to the heterodimerisation process, and Orf16, which has a noncatalytic, auxiliary role. The biosynthesis of tetronates such as kitaniitetronin A 22 from Kitasatospora niigatensis DSM 44781 has been elucidated and shown to involve a partially functional nonribosomal peptide synthase (NRPS) and an intact PKS, which facilitate the condensation of ketonic acid and malonyl-CoA-like extender units.…”