Abstract
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.