Elucidating the Iterative Elongation Mechanism in a Type III Polyketide Synthase

Type III polyketide synthases (PKSs) have a much simpler
three-dimensional
architecture compared with their type I and type II counterparts,
yet they catalyze iterative polyketide elongation to generate a myriad
of products in plants, fungi, and eubacteria. Despite this mechanistic
complexity occurring within a single active site, the mechanism by
which type III PKSs stabilize and direct their highly reactive keto
and enolate intermediates has yet to be fully understood. Here, we
report the synthesis and deployment of stable polyketone CoA analogues
for each putative intermediate involved in the biphenyl synthase (BIS)
mechanism together with three high-resolution crystal structures of
each in complex with BIS from Malus domestica. This set of structures reveals key mechanistic features that control
the number of iterative elongation steps and that shape the static
architectural features responsible for organization of a water-mediated
hydrogen bonding network necessary for termination of the elongation
reaction by an intramolecular aldol cyclization and production of
the 3,5-dihydroxybiphenyl BIS product. Elucidating these protein–substrate
interactions provides a foundation for using polyketone CoA analogues
to further unravel the control mechanisms of PKS catalysis and gain
the insight necessary for predictive engineering of these enzymes.