Towards understanding oxygen heterocycle-forming biocatalysts: a selectivity study of the pyran synthase PedPS7.

Intramolecular oxa-Michael addition-catalysing cyclases are widespread in polyketide biosynthetic pathways. Although they have significant potential in biotechnology and chemoenzymatic synthesis of chiral heterocycles, they have only scarcely been studied. Here, we present detailed investigations on the selectivity profile of the pyran synthase PedPS7 showing that it combines broad substrate tolerance with high selectivity for the formation of up to two new stereocentres and relaxed precursor stereoisomer discrimination. Two of the four possible tetrahydropyran stereoisomers are reliably accessible by this enzyme. The results indicate fundamental differences between the individual subtypes of intramolecular oxa-Michael addition-catalysing cyclases.

Insights into the Dual Activity of a Bifunctional Dehydratase-Cyclase Domain.

Oxygen-containing heterocycles are a common structural motif in polyketide natural products and contribute significantly to their biological activity. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratase (DH) and pyran-forming cyclase in ambruticin biosynthesis. AmbDH3 is similar to monofunctional DH domains, using H51 and D215 for dehydration. V173 was confirmed as a diagnostic residue for cyclization activity by a mutational study and enzymatic in vitro experiments. Similar motifs were observed in the seemingly monofunctional AmbDH2, which also shows an unexpected cyclase activity. Our results pave the way for mining of hidden cyclases in biosynthetic pathways. They also open interesting prospects for the generation of novel biocatalysts for chemoenzymatic synthesis and pyran-polyketides by combinatorial biosynthesis.

Cross-linking of a polyketide synthase domain leads to a recyclable biocatalyst for chiral oxygen heterocycle synthesis.

The potential of polyketide synthase (PKS) domains for chemoenzymatic synthesis can often not be tapped due to their low stability and activity in vitro. In this proof-of-principle study, the immobilisation of the heterocycle-forming PKS domain AmbDH3 as a cross-linked enzyme aggregate (CLEA) is described. The AmbDH3-CLEA showed good activity recovery, stability and recyclability. Repetitive reactions on the semi-preparative scale were performed with high conversion and isolated yield. Similar to that observed for the free enzyme, the aggregate retained substrate tolerance and the ability for kinetic resolution. This first example of a successful enzymatic PKS domain immobilisation demonstrates that cross-linking can in principle be applied to this type of enzyme to increase its applicability for chemoenzymatic synthesis.