Polycyclic tetramate macrolactams
(PTMs) are a class of structurally
complex hybrid polyketide-nonribosomal peptide (PK-NRP) natural products
produced by diverse bacteria. Several PTMs display pharmaceutically
interesting bioactivities, and the early stages of PTM biosynthesis
involving polyketide synthase (PKS) and nonribosomal peptide synthetase
(NRPS) enzymology are well studied. However, the timing and mechanisms
of post PKS-NRPS oxidations by P450 monooxygenases encoded in PTM
biosynthetic gene clusters (BGCs) remain poorly characterized. Here
we demonstrate that CftA, encoded in clifednamide-type PTM BGCs, is
a multifunctional P450 monooxygenase capable of converting the C29–C30
ethyl side chain of ikarugamycin to either a C29–C30 methyl
ketone or a C29–C30 hydroxymethyl ketone through C–H
bond activation, resulting in the formation of clifednamide A or clifednamide
C, respectively. We also report the complete structure of clifednamide
C solved via multidimensional NMR (COSY, HSQC, HMBC, NOESY, and TOCSY)
using material purified from an engineered Streptomyces strain optimized for production. Finally, the in vitro reconstitution of recombinant CftA catalytic activity revealed the
oxidation cascade for sequential conversion of ikarugamycin to clifednamide
A and clifednamide C. Our findings confirm prior genetics-based predictions
on the origins of clifednamide complexity via P450s encoded in PTM
BGCs and place CftA into a growing group of multifunctional P450s
that tailor PTM natural products through late-stage regioselective
C–H bond activation.