The structural organization of substrate loading in iterative polyketide synthases.

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

Examining Heterodimerization by Aryl C-N Coupling in Dynemicin Biosynthesis.

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 ß-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.

Dynemicin A Derivatives as Potential Cancer Chemotherapeutics by Mutasynthesis.

The enediyne antitumor antibiotics have remarkable structures and exhibit potent DNA cleavage properties that have inspired continued interest as cancer therapeutics. Their complex structures and high reactivity, however, pose formidable challenges to their production and development in the clinic. We report here proof-of-concept studies using a mutasynthesis strategy to combine chemical synthesis of select modifications to a key iodoanthracene-γ-thiolactone intermediate in the biosynthesis of dynemicin A and all other known anthraquinone-fused enediynes (AFEs). By chemical complementation of a mutant bacterial producer that is incapable of synthesizing this essential building block, we show that derivatives of dynemicin can be prepared substituted in the A-ring of the anthraquinone motif. In the absence of competition from native production of this intermediate, the most efficient utilization of these externally-supplied structural analogues for precursor-directed biosynthesis becomes possible. To achieve this goal, we describe the required Δorf15 blocked mutant and a general synthetic route to a library of iodoanthracene structural variants. Their successful incorporation opens the door to enhancing DNA binding and tuning the bioreductive activation of the modified enediynes for DNA cleavage.