FD-891

GfsF is a multifunctional P450 monooxygenase that catalyzes epoxidation and subsequent hydroxylation in the biosynthesis of macrolide polyketide FD-891. Here, we describe the biochemical and structural analysis of GfsF. To obtain the structural basis of a dual-function reaction, we determined the crystal structure of ligand-free GfsF, which revealed GfsF to have a predominantly hydrophobic substrate binding pocket. The docking models, in conjunction with the results of the enzymatic assay with substrate analogues and site-directed mutagenesis suggested two distinct substrate binding modes for epoxidation and hydroxylation reactions, which explained how GfsF regulates the order of two oxidative reactions. These findings provide new insights into the reaction mechanism of multifunctional P450 monooxygenases.

A concise and unified strategy for the synthesis of C1-C18 macrolactone fragments of FD-891 and FD-892 as well as their analogues is reported. The strategy includes a stereoselective vinylogous Mukaiyama aldol reaction (VMAR) using chiral silyl ketene N,O-acetal to construct C6-C7 stereocenters, an E-selective ring closing metathesis to construct a C12-C13 olefin, and stereodivergent construction of a C8-C9 epoxide.

To isolate a key polyketide biosynthetic intermediate for the 16-membered macrolide FD-891 (1), we inactivated two biosynthetic genes coding for post-polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD-892 (2), which lacks the epoxide moiety at C8-C9, the hydroxy group at C10, and the O-methyl group at O-25 of FD-891, was isolated from the gfsF/gfsG double-knockout mutant. In addition, 25-O-methyl-FD-892 (3) and 25-O-demethyl-FD-891 (4) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8-C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1, respectively. These results suggest that a parallel post-PKS modification mechanism is involved in FD-891 biosynthesis.