Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-11, Vol.107 (46), p.19778-19783
• Main Authors: Morita, Hiroyuki, Wanibuchi, Kiyofumi, Nii, Hirohiko, Kato, Ryohei, Sugio, Shigetoshi, Abe, Ikuro, Croteau, Rodney B.
• Format: Article
• Language: English
• Subjects: Binding sites; Biochemistry - methods; Biological Sciences; Catalytic Domain; Chalconoids; Chromatography, High Pressure Liquid; Condensation; Crystal structure; Crystallography, X-Ray; Diarylheptanoids - chemistry; Diarylheptanoids - metabolism; Electrons; Enzymes; Hydrogen Bonding; Hydrogen bonds; Ligases - chemistry; Ligases - metabolism; Models, Molecular; Molecular structure; Molecules; Monomers; Mutagenesis - genetics; Mutation; Oryza - enzymology; Polyketides; Rice; Scaffolds; Structure-Activity Relationship; Substrate specificity; Thiolester Hydrolases - chemistry; Thiolester Hydrolases - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1011499107

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C₆-C₇-C₆ diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C₆-C₃ coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H₂O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C₆-C₇-C₆ scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes.