Structural control of polyketide formation in plant-specific polyketide synthases

Background: Polyketide synthases (PKSs) generate molecular diversity by uti lizing different starter molecules and by controlling the final length of t he polyketide. Although exploitation of this mechanistic variability has pr oduced novel polyketides, the structural foundation of this versatility is unclear. Plant-specific PKSs are essential for the biosynthesis of anti-mic robial phytoalexins, anthocyanin floral pigments, and inducers of Rhizobium nodulation genes. 2-Pyrone synthase (2-PS) and chalcone synthase (CHS) are plant-specific PKSs that share 74% amino acid sequence identity. 2-PS form s the triketide methylpyrone from an acetyl-CoA starter molecule and two ma lonyl-CoAs. CHS uses a p-coumaroyl-CoA starter molecule and three malonyl-C oAs to produce the tetraketide chalcone. Our goal was to elucidate the mole cular basis of starter molecule selectivity and control of polyketide lengt h in this class of PKS. Results: The 2.05 Angstrom resolution crystal structure of 2-PS complexed w ith the reaction intermediate acetoacetyl-CoA was determined by molecular r eplacement. 2-PS and CHS share a common three-dimensional fold, a set of co nserved catalytic residues, and similar CoA binding sites. However, the act ive site cavity of 2-PS is smaller than the cavity in CHS. Of the 28 residu es lining the 2-PS initiation/elongation cavity, four positions vary in CHS , Point mutations at three of these positions in CHS (T197L, G256L, and S33 8I) altered product formation. Combining these mutations in a CHS triple mu tant (T197L/G256L/S3381) yielded an enzyme that was functionally identical to 2-PS. Conclusions: Structural and functional characterization of 2-PS together wi th generation of a CHS mutant with an initiation/elongation cavity analogou s to 2-PS demonstrates that cavity volume influences the choice of starter molecule and controls the final length of the polyketide. These results pro vide a structural basis for control of polyketide length in other PKSs, and suggest strategies for further increasing the scope of polyketide biosynth etic diversity.