The Modular Polyketide Synthase Story: The Michael Edition

By Ian Smith

Paper: Vinylogous chain branching catalysed by a dedicated polyketide synthase module
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature12588.html

Type I PKS for assembly of the rhizoxin backbone and structure of rhizoxin D. ACP, acyl carrier protein; AT, acyl transferase; B, branching domain; DH, dehydratase; KR, ketoreductase; KS, ketosynthase. b, Possible course of chain-branching reaction highlighting two different lactonization routes.
Type I PKS for assembly of the rhizoxin backbone and structure of rhizoxin D. ACP, acyl carrier protein; AT, acyl transferase; B, branching domain; DH, dehydratase; KR, ketoreductase; KS, ketosynthase. b, Possible course of chain-branching reaction highlighting two different lactonization routes.

Creating complex naturally-occurring compounds biosynthetically, by mimicking prokaryotic metabolism, is opening doors to the creation of useful clinical therapeutics. For instance, modular polyketide synthases (PKSs), thus far, have been exclusively studied for their therapeutic potential and their ability to synthesize long linear polyketides. To date, the known PKS modules involve the catalysis of a keto-synthase(KS)-mediated Claisen condensation involving an ACP-anchored malonyl and KS-tethered acyl unit. Although this known module for the linear polymerization of polyketides can yield biosynthetic engineering capabilities, a new study by Bretschneider et al. (Nature. 2013, 502, published online October 3, DOI: 10.1038/nature12588)indicates a new PKS module, as demonstrated by the biosynthesis of a promising anti-tumoral agent called rhizoxin in Burkholderia rhizoxinica.
The new PKS module incorporates the typical ACP and KS domain, but this module also requires the use of a branching domain (B) that structurally assists the KS domain to carry out an unprecedented vinylogous Michael addition of the malonyl unit to the KS-bound acyl unit. This Michael addition results in polyketide branching and δ -lactone ring formation that is essential for rhizoxin’s anti-mitotic properties. By carrying out the PKS module in vitro, the authors could effectively characterize the ACP adduct containing the δ -lactone ring, via MALDI-TOF, and the liberated lactone, via LC-MS. With the lactone identified, the authors indicate what catalytic residues of B and KS domains, via X-ray crystallography, are structurally important for the catalytic functionality of the module. The authors determine that KS and B domains cannot catalyze the Michael addition independently and a point mutation of B’s catalytically relevant aspartate has no effect on branching activity. With structural insight being insufficient for determining this PKS’s module, the authors use the NMR analysis of the in vitro assay with 13C-labelled malonyl and gel electrophoresis of the in vitro assay with a 5-deoxy acyl unit to identify that δ-lactone formation involves the attack of the acyl’s C5 hydroxyl group at the acyl’s C1 cabonyl, resulting in the malonyl unit as a side chain. The implications of this novel module include broadening the repertoire of natural PKS reactions for biosynthetic engineering of new polyketides and providing the biosynthetic utility to create therapeutic analogues. Also, this Michael Edition of modular PKS can provide insight into similar and unknown mechanisms for the production of glutarimide-containing antibiotics.