Structural and biochemical elucidation of mechanism for decarboxylative condensation of beta-keto acid by curcumin synthase

Abstract

The typical reaction catalyzed by type III polyketide synthases (PKSs) is a decarboxylative condensation between acyl-CoA (starter substrate) and malonyl-CoA (extender substrate). In contrast, curcumin synthase 1 (CURS1), which catalyzes curcumin synthesis by condensing feruloyl-CoA with a diketide-CoA, uses a β-keto acid (which is derived from diketide-CoA) as an extender substrate. Here, we determined the crystal structure of CURS1 at 2.32 Å resolution. The overall structure of CURS1 was very similar to the reported structures of type III PKSs and exhibited the αβαβα fold. However, CURS1 had a unique hydrophobic cavity in the CoA-binding tunnel. Replacement of Gly-211 with Phe greatly reduced the enzyme activity. The crystal structure of the G211F mutant (at 2.5 Å resolution) revealed that the side chain of Phe-211 occupied the hydrophobic cavity. Biochemical studies demonstrated that CURS1 catalyzes the decarboxylative condensation of a β-keto acid using a mechanism identical to that for normal decarboxylative condensation of malonyl-CoA by typical type III PKSs. Furthermore, the extender substrate specificity of CURS1 suggested that hydrophobic interaction between CURS1 and a β-keto acid may be important for CURS1 to use an extender substrate lacking the CoA moiety. From these results and a modeling study on substrate binding, we concluded that the hydrophobic cavity is responsible for the hydrophobic interaction between CURS1 and a β-keto acid, and this hydrophobic interaction enables the β-keto acid moiety to access the catalytic center of CURS1 efficiently.

FIGURE 1.

Reactions catalyzed by chalcone synthase (A) and by curcumin synthase 1 (B and C). A, CHS catalyzes condensation of p-coumaroyl-CoA and three molecules of malonyl-CoA to synthesize the tetraketide intermediate. The resulting tetraketide intermediate is further cyclized by CHS and converted to naingenin chalcone. B, CURS1 catalyzes the hydrolysis of diketide-CoA to yield a β-keto acid (ii) and decarboxylative condensation of the β-keto acid with feruloyl-CoA to yield curcumin (iii). CURS1 scarcely catalyzes the formation of diketide-CoA from feruloyl-CoA and malonyl-CoA (i). In C. longa, this reaction is primarily catalyzed by a diketide-CoA synthase. C, for analysis of the activity of CURS1, cinnamoylferuloylmethane (curcuminoid) formation from feruloyl-CoA with cinnamoyldiketide-NAC (an analog of diketide-CoA) or 3-oxo-5-phenyl-4-pentenoic acid (a β-keto acid) was examined.

FIGURE 2.

Overall structure of CURS1 (A and B) and amino acid alignment of CURS1 with other type III PKSs (C). A, CURS1 dimer exhibited the αβαβα fold, similar to other type III PKSs. One monomer is highlighted in green and the other in dark blue. The side chains of Cys-164, His-303, Asn-336, and Met-137 are depicted. B, overall structure of a CURS1 monomer (green) is superimposed on that of CHS (magenta). Both enzymes have a very similar structure. C, catalytic triad (Cys-164, His-303, and Asn-336 in CURS1) is highlighted in orange and two phenylalanines (Phe-215 and Phe-265 in CURS1) that are called “gatekeepers” are highlighted in blue. ClCURS1, CURS1 (curcumin synthase 1) from C. longa (BAH56226); OsCUS, curcuminoid synthase from O. sativa (AK109558); ClDCS, diketide-CoA synthase from C. longa (BAH56225); MsCHS, CHS from Medicago sativa (AAA02824); RhBAS, BAS from Rheum palmatum (AAK82824).

FIGURE 3.

Wall-eye stereo view of the structure of active site pocket of CURS1 (chain A). A, white sticks indicate the side chains of the amino acid residues around the active site pocket of CURS1. The malonic acid observed in the active site pocket is shown as green sticks. The cyan sticks indicate the CoA (left) and naringenin (right) bound with CHS. They are superimposed with CURS1 for the comparison of the active site pockets between CURS1 and CHS. B, side chains of the amino acid residues around the active site pocket of CHS (orange) are superimposed on those of CURS1 (white).

FIGURE 4.

Curcuminoid synthesis activity of the wild-type and mutant CURS1 enzymes from feruloyl-CoA with cinnamoyldiketide-NAC (gray bars) or 3-oxo-5-phenyl-4-pentenoic acid (β-keto acid) (white bars).

FIGURE 5.

Orientation of the phenyl group generated by the G211F mutation. The white sticks indicate the side chains of the amino acid residues around the active site pocket of wild-type CURS1. Magenta sticks indicate the phenyl group generated by the G211F mutation. Green sticks indicate side chains of the other amino acid residues of CURS1 G211F.

FIGURE 6.

Model for the binding of a feruloyl moiety and a β-keto acid in the active site pocket of CURS1. A, native structure of the active site pocket of CURS1. B, most reliable model structure. A feruloyl moiety and a β-keto acid are colored magenta and green, respectively.

FIGURE 7.

Proposed mechanism of the reaction catalyzed by CURS1. First, the feruloyl moiety of feruloyl-CoA is transferred to the catalytic Cys of CURS1. Then a diketide-CoA interacts with CURS1 and is hydrolyzed to a β-keto acid. The resulting β-keto acid interacts with the catalytic center with the help of the hydrophobic cavity located around Phe-265 and is decarboxylated to form an active anion. The active anion attacks the carbon of ketone of the feruloyl moiety to form curcumin.