1. Life Sciences Institute,
2. Department of Medicinal Chemistry,
3. Department of Chemistry,
4. Department of Biological Chemistry,
5. Department of Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan 48109, USA
6. Department of Chemistry and Center for Chemical Methodologies & Library Development, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA
7. Scripps Institution of Oceanography & Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, USA
8. These authors contributed equally to this work.

Correspondence to: David H. Sherman^1,2,3,5 Correspondence and requests for materials should be addressed to D.H.S. (Email: davidhs@umich.edu).

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism¹. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a -branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide -branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH₁s), decarboxylases (ECH₂s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide -branching pathway was modified by introduction of a -chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), -ketoglutarate-dependent enzyme². In a divergent scheme, Cur ECH₂ was found to catalyse formation of the , enoyl thioester, whereas Jam ECH₂ formed a vinyl chloride moiety by selectively generating the corresponding , enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH₂ instead of the canonical , C = C saturation reaction. Thus, the combination of chlorination and polyketide -branching, coupled with mechanistic diversification of ECH₂ and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.

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