Metamorphic enzyme assembly in polyketide diversification

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism. 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 Β-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 Β-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH"1s), decarboxylases (ECH"2s) 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 Β-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), α-ketoglutarate-dependent enzyme. In a divergent scheme, Cur ECH"2 was found to catalyse formation of the α,Β enoyl thioester, whereas Jam ECH"2 formed a vinyl chloride moiety by selectively generating the corresponding Β, 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 ECH"2 instead of the canonical α,Β C ≤ C saturation reaction. Thus, the combination of chlorination and polyketide Β-branching, coupled with mechanistic diversification of ECH"2 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.