Structural control of polyketide formation in plant-specific polyketide synthases

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Polyketide synthases (PKSs) are crucial enzymes in natural product biosynthesis, generating molecular diversity through variable starter molecules and controlled polyketide chain length.
• Plant-specific PKSs synthesize vital compounds like phytoalexins and floral pigments, with 2-pyrone synthase (2-PS) and chalcone synthase (CHS) exemplifying this class.
• Understanding the structural basis for PKS versatility, particularly starter molecule selectivity and chain length control, remains a key challenge.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying starter molecule selectivity and polyketide chain length determination in plant-specific PKSs.
• To investigate the structural differences between 2-pyrone synthase (2-PS) and chalcone synthase (CHS) that contribute to their distinct biochemical activities.

Main Methods:

• Determined the crystal structure of 2-pyrone synthase (2-PS) at 2.05 Å resolution using molecular replacement, complexed with the reaction intermediate acetoacetyl-CoA.
• Performed site-directed mutagenesis on chalcone synthase (CHS) by altering residues within the active site cavity.
• Assessed the functional impact of mutations on CHS activity and product formation.

Main Results:

• 2-PS and CHS share a conserved three-dimensional fold, catalytic residues, and CoA binding sites, but 2-PS possesses a smaller active site cavity.
• Four residues lining the active site cavity differ between 2-PS and CHS.
• Mutagenesis of three specific residues (T197L, G256L, S338I) in CHS altered its product profile, with a triple mutant exhibiting 2-PS-like functionality.

Conclusions:

• The volume of the active site cavity in plant PKSs is a critical determinant of starter molecule selection and polyketide chain length.
• Structural insights into 2-PS and engineered CHS provide a foundation for understanding PKS versatility.
• These findings suggest strategies for engineering PKSs to expand the diversity of polyketide natural products.