A structure-based mechanism for benzalacetone synthase from Rheum palmatum

Abstract

Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 A resolution. In addition, we solved the crystal structure of the wild-type enzyme, in which a monoketide coumarate intermediate is covalently bound to the catalytic cysteine residue, at 1.6 A resolution. This is the first direct evidence that type III PKS utilizes the cysteine as the nucleophile and as the attachment site for the polyketide intermediate. The crystal structures revealed that BAS utilizes an alternative, novel active-site pocket for locking the aromatic moiety of the coumarate, instead of the chalcone synthase's coumaroyl-binding pocket, which is lost in the active-site of the wild-type enzyme and restored in the I207L/L208F mutant. Furthermore, the crystal structures indicated the presence of a putative nucleophilic water molecule which forms hydrogen bond networks with the Cys-His-Asn catalytic triad. This suggested that BAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

Fig. 1.

Enzyme reaction and active-site structure of R. palmatum BAS. (A) Proposed mechanism for the formation of benzalacetone by BAS. The active-site structures of wild-type BAS apo (B), M. sativa CHS (C), BAS I207L/L208F mutant (D), and wild-type BAS with the covalently bound coumarate (E). The coumarate and naringenin molecules are shown as blue and green stick models, respectively. The water molecule and the hydrogen bonds are indicated with light-blue spheres and dotted lines, respectively. The F_o-F_c density map of the monoketide intermediate covalently bound to the catalytic Cys157 (F) in monomer A, and in monomer B of wild-type BAS, countered at 2.0 s (G).

Fig. 2.

Surface and schematic representations of the active-site cavities of wild-type BAS (A and D), M. sativa CHS (B and E) , and BAS I207L/L208F mutant (C and F). The bottoms of the “coumaroyl-binding pocket” are highlighted as purple surfaces. The covalently bound coumarate, the water molecules and the hydrogen bonds are colored as in Fig. 1.

Fig. 3.

Proposed mechanism for the BAS enzyme reaction. The active-site structures of the apo (A) and the coumarate-bound BAS (B), respectively. Three-dimensional model for the diketide intermediate covalently bound to the catalytic cysteine. The putative nucleophilic water molecule is shown as a light-blue sphere. The dotted lines indicate the hydrogen bond (C). Proposed mechanism for the enzyme catalyzed hydrolysis and decarboxylation reaction (D).