A Long-Range Acting Dehydratase Domain as the Missing Link for C17-Dehydration in Iso-Migrastatin Biosynthesis

Abstract

The dehydratase domains (DHs) of the iso-migrastatin (iso-MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module-6, -9, -10 of MgsF (i.e., DH6, DH9, DH10) and module-11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso-MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β-hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long-range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.

Keywords: biosynthesis; dehydratase; iso-migrastatin; natural products; polyketides.

Figure 1

Selected glutarimide-containing polyketide family of natural products with or without a hydroxy group at the C17-positions (1, 2, 6, 7) or equivalents (3, 4, 5).

Figure 2

Comparative analysis of the iso-MGS and LTM PKSs identifying the “missing” dehydratase activity for 1 biosynthesis. The iso-MGS and LTM PKSs produce 8 and 9 as the nascent polyketide intermediates that are transformed to 1 and 2 by MgsIJK and LtmK, respectively, and four DHs are predicted according to type I PKS collinear model for 8 and 9 biosynthesis. The “missing” dehydratase activity for C16–C17 double bond formation in 8 is shown in this study to be provided by DH10 in a long-range acting manner. TE, thioesterase.

Figure 3

Systematic inactivation of DH6, DH9, DH10, and DH11 of iso-MGS PKS allowed functional assignment of their roles in biosynthesis of 1. (A) Structures of the metabolites (using same numbering as 1 with the varying groups) characterized from the DH mutants. (B) Metabolite profiles of selected DH mutants (II–VI) in comparison with the control (I)

Figure 4

In vitro assays demonstrating MgsF-DH10 as a dehydratase acting in a long-range acting manner. HPLC profiles of MgsF-DH10 catalyzed (ii) dehydration in comparison with (i) the negative control using SNAC thioesters (A) 19, (B) 21, and (C) 23 mimicking the growing polyketide intermediates tethered to the ACPs in PKS module-4, -10, and -11, respectively.