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.