Structure of a modular polyketide synthase reducing region.

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Ketoreductase Domain Dysfunction Expands Chemodiversity: Malyngamide Biosynthesis in the Cyanobacterium Okeania hirsuta.

Dozens of type A malyngamides, principally identified by a decorated six-membered cyclohexanone headgroup and methoxylated lyngbic acid tail, have been isolated over several decades. Their environmental sources include macro- and microbiotic organisms, including sea hares, red alga, and cyanobacterial assemblages, but the true producing organism has remained enigmatic. Many type A analogues display potent bioactivity in human-health related assays, spurring an interest in this molecular class and its biosynthetic pathway. Here, we present the discovery of the type A malyngamide biosynthetic pathway in the first sequenced genome of the cyanobacterial genus Okeania. Bioinformatic analysis of two cultured Okeania genome assemblies identified 62 and 68 kb polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) pathways with unusual loading and termination genes. NMR data of malyngamide C acetate derived from 13C-substrate-fed cultures provided evidence that an intact octanoate moiety is transferred to the first KS module via a LipM homologue originally associated with lipoic acid metabolism and implicated an inactive ketoreductase (KR0) as critical for six-membered ring formation, a hallmark of the malyngamide family. Phylogenetic analysis and homology modeling of the penultimate KR0 domain inferred structural cofactor binding and active site alterations as contributory to domain dysfunction, which was confirmed by recombinant protein expression and NADPH binding assay. The carbonyl retained from this KR0 ultimately enables an intramolecular Knoevenagel condensation to form the characteristic cyclohexanone ring. Understanding this critical step allows assignment of a biosynthetic model for all type A malyngamides, whereby well-characterized tailoring modifications explain the surprising proliferation and diversity of analogues.