Question. Various bacterial tropone natural products such as the antibiotic sulphur-containing tropodithietic acid (TDA) or (hydroxy)tropolones adopt crucial roles in symbiotic interactions with eukaryotic hosts. Their biosynthesis relies on an unusual intertwining of primary and secondary metabolism, in which the initial steps are shared for the various tropones and rely on enzymes from phenylacetic acid (paa) catabolism. In this pathway, a distinct reactive open-chain aldehyde intermediate is formed, which is usually further degraded to central metabolites. In the case of tropone biosynthesis, however, this compound is instead cyclized to the characteristic tropone scaffold. Yet, it remains largely unknown how tailoring enzymes in certain bacteria further convert the tropone scaffold into the mature natural products.
Methods. We employed in vitro reconstitution of diverse tropone biosynthetic pathways from Gram-negative and Gram-positive bacteria using heterologously produced enzymes from paa catabolism as well as enzymes encoded by different tropone biosynthetic gene clusters. Key enzymes were furthermore characterized mechanistically by enzyme assays and structurally by X-ray crystallography to obtain insights into their reaction mechanisms.
Results. We unravelled an unexpected mechanistic diversity for tropone functionalization involving distinct key flavoenzymes, which drastically modify the tropone scaffolds in the late-stage biosynthesis of TDA from Gram-negative Phaeobacter sp. and 3,7-dihydroxytropolone from Streptomyces sp., respectively. In both cases, these enzymes mediate several unanticipated reactions en route to the mature natural products such as a so far unique flavoenzyme-catalyzed dioxygenation in TDA biosynthesis involving oxygenolytic CoA-thioester cleavage followed by a regioselective ring epoxidation.
Conclusions. The biosynthesis of 3,7-dihydroxytropolone could be completely reconstituted and functions assigned to all involved enzymes. Similarly TDA biosynthesis was scrutinized, allowing for the discovery of a novel flavoprotein dioxygenase archetype, while final pathway steps involving the sulphur incorporation are subject to further investigation.