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 Å 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 Å 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 activesite 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.biosynthesis | enzyme | polyketide

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

“…The catalytic triad of Cys174, His316, and Asn349 is buried deep within each monomer and sits at the intersection of a traditional 16-Å-long CoA binding tunnel and a large internal cavity, in a location and orientation very similar to those of the other plant type III PKSs. A structure-based similarity search using the Dali program (13) suggested that the overall structure of CUS is highly homologous to those of the previously reported plant type III PKSs (rmsds 1.0-1.2 Å and 2.0-2.2 Å for plant and bacterial type III PKSs, respectively), in which the most closely related structural homologue is that of Rheum palmatum benzalacetone synthase (BAS) (PDB entry 3A5Q, Z score ¼ 57.2, rmsd of 1.0 Å over 360 residues aligned with a sequence identity of 53%) (14). R. palmatum BAS produces the diketide benzalacetone by the one-step condensation of 4-coumaroyl-CoA and malonyl-CoA through the formation of 4-coumaroyldiketide acid (Fig.…”

“…2 C and D). The coumaroyl binding pocket is also absent in the active-site cavity of R. palmatum BAS, but in this case, the contraction is caused by the simultaneous substitution of the CHS's conserved Thr132 and Phe215 with Leu, along with the conformational change of Ser338, and as a result, the enzyme catalyzes only one condensation with malonyl-CoA to produce the diketide benzalacetone (14).…”

“…Our previous crystallographic studies on R. palmatum BAS revealed that BAS utilizes a nucleophilic water molecule, which forms hydrogen bond networks with the Cys-His-Asn catalytic triad, for the thioester bond cleavage of the diketide intermediate bound to the catalytic Cys164, to produce the coumaroyldiketide acid intermediate (Figs. 1G and 2G) (14). On the other hand, crystallographic studies of P. sylvestris STS revealed the so-called "aldol-switch" hydrogen bond network, including Thr132 and a nucleophilic water molecule neighboring the catalytic Cys164 at the active-site center of STS (numbering in M. sativa CHS) (Figs.…”

“…It is possible that the diketide acid is produced by nonenzymatic, spontaneous hydrolysis of the 4-coumaroyldiketide-CoA, once it is formed and released from the enzyme. However, another possibility is the enzymatic formation of the diketide acid by the thioester bond cleavage of the enzyme-bound intermediate, as in the case of R. palmatum BAS (14). Our previous crystallographic studies on R. palmatum BAS revealed that BAS utilizes a nucleophilic water molecule, which forms hydrogen bond networks with the Cys-His-Asn catalytic triad, for the thioester bond cleavage of the diketide intermediate bound to the catalytic Cys164, to produce the coumaroyldiketide acid intermediate (Figs.…”

Type III polyketide synthases in natural product biosynthesis

“…The crystal structure of BAS also indicated the presence of a putative nucleophilic water molecule that forms hydrogen bond networks with the Cys-His-Asn catalytic triad. Additionally, BAS uses novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone (22).…”