How <i>cis</i>-Acyltransferase Assembly-Line Ketosynthases Gatekeep for Processed Polyketide Intermediates

With
the redefinition of polyketide synthase (PKS) modules, a new
appreciation of their most downstream domain, the ketosynthase (KS),
is emerging. In addition to performing its well-established role of
generating a carbon–carbon bond between an acyl-CoA building
block and a growing polyketide, it may gatekeep against incompletely
processed intermediates. Here, we investigate 739 KSs from 92 primarily
actinomycete, cis-acyltransferase assembly lines.
When KSs were separated into 16 families based on the chemistries
at the α- and β-carbons of their polyketide substrates,
a comparison of 32 substrate tunnel residues revealed unique sequence
fingerprints. Surprisingly, additional fingerprints were detected
when the chemistry at the γ-carbon was considered. Representative
KSs were modeled bound to their natural polyketide substrates to better
understand observed patterns, such as the substitution of a tryptophan
by a smaller residue to accommodate an l-α-methyl group
or the substitution of four smaller residues by larger ones to make
better contact with a primer unit or diketide. Mutagenesis of a conserved
glutamine in a KS within a model triketide synthase indicates that
the substrate tunnel is sensitive to alteration and that engineering
this KS to accept unnatural substrates may require several mutations.