Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa.
Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C(6)-C(7)-C(6) 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 C(6)-C(3) 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-H(2)O-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 C(6)-C(7)-C(6) 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.
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Hiroyuki
H
Morita
Kiyofumi
K
Wanibuchi
Hirohiko
H
Nii
Ryohei
R
Kato
Shigetoshi
S
Sugio
Ikuro
I
Abe
D001671:Biochemistry / Q000379:methods*; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D036381:Diarylheptanoids / Q000737:chemistry* / Q000378:metabolism*; D004583:Electrons; D006860:Hydrogen Bonding; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D012275:Oryza / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000378:metabolism
D036381:Diarylheptanoids; D013869:Thiolester Hydrolases; D008025:Ligases