Uncovering the Parallel Biosynthetic Pathways of the Cyclohexanone and Phenol Rings in Cycloheximide and Actiphenol by Tailoring Redox Enzymes

The eukaryotic translation inhibitor cycloheximide (CHX) and its analogue actiphenol (APN) feature a glutarimide moiety and a six-membered carbocyclic ring system. The biosynthesis of CHX and APN is not yet fully understood, particularly with respect to the mechanism of formation of the fully reduced cyclohexanone ring in CHX and the aromatic phenol ring in APN. In this work, a combination of gene inactivation, chemical synthesis, and in vitro biochemical experiments highlighted an ensemble of three tailoring redox enzymes as being responsible for the biosynthesis of the six-membered carbocyclic ring systems. Specifically, two redox enzymes (ChxJ and ChxI) alone can generate an active intermediate that undergoes a cascade of non-enzymatic transformations to create APN, while a reductive enzyme (ChxG) acts as a gatekeeper, directing the same intermediate down a different pathway toward CHX. Finally, the full nature of each biosynthetic pathway was established in detail, including the formation mechanisms of six-membered carbocyclic rings.