Document Type


Journal/Book Title

Proceedings of the National Academy of Sciences of the United States of America

Publication Date



Resorcylic acid lactones (RAL) and dihydroxyphenylacetic acid lactones (DAL) represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS (hrPKS) whose product is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates towards defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the DAL dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal versus the bacterial folding mode difference is portable upon creating hybrid enzymes, and structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations, and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first ring cyclizations will facilitate efforts towards the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.


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