Structural elucidation of chalcone reductase and implications for deoxychalcone biosynthesis

Abstract

4,2',4',6'-Tetrahydroxychalcone (chalcone) and 4,2',4'-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/keto-reductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase.

Fig. 1. CHS/CHR reaction pathway

Left panel, the CHS-catalyzed pathway to chalcone. Right panel, arrows indicate reaction steps where CHR could catalyze ketoreduction and commit CHS intermediates to deoxychalcone biosynthesis.

Fig. 2. Evolutionary tree showing representative enzymes of AKR families 1–5 (48)

Purple and green represent enzymes of the AKR4 family. AKR4 family enzymes of largely undefined function are highlighted in purple; CHR and COR enzymes from several species are highlighted in green.

Fig. 3. The trione ring of coumaryl-trione adopts a modified boat conformation after MM2 minimization in CHEM3D Ultra (Cambridge Soft Corporation)

Carbons (C1), (C2), (C4), and (C5) are labeled as such to facilitate discussion, yet because of the prochirality of C6 and the unknown stereochemistry of the intramolecular cyclization reaction catalyzed by CHS, the precise origin of these carbons relative to the linear tetraketide-CoA is unknown, unlike carbons C3 and C6.

Fig. 4. Ribbon representations of CHR

A, the (α/β)₈-barrel helices are colored in red, and β-strands are blue. The two additional α-helices known to pack against the outer barrel in AKRs are purple. NADP⁺ is shown in gold in ball-and-stick representation. B, ribbon representation of loop structures in CHR. NADP⁺ is shown in gold. C, overlays of CHR, 3α-hydroxy-steroid dehydrogenase (1J96), and aldose reductase (1EF3) illustrate divergence in CHR loop structure.

Fig. 5. Stick representation of NADP⁺ bound in the CHR active site

Hydrogen bonds are represented by small green spheres. Half-colored bonds represent individual atoms with red, gray, blue, and magenta, representing oxygen, carbon, nitrogen, and phosphorus atoms, respectively. Carbon atoms are colored gold for NADP⁺ only.

Fig. 6. CHR active site

NADP⁺ is shown in gold, and docked coumaryl-trione is cyan. Residues of the catalytic tetrad are labeled in pink. Proposed hydrogen bonding patterns are shown in small green and black spheres. Half-colored bonds represent individual atoms with red, gray, blue, and magenta representing oxygen, carbon, nitrogen, and phosphorus atoms, respectively. Carbon atoms are colored gold for NADP⁺ only. Carbon atoms are colored green for coumaryl-trione only.

Fig. 7

The three-dimensional architecture of the trione ring of coumaryl-trione (blue) is very similar to the glucose ring of UDP-glucose (yellow), another AKR family enzyme substrate.

Fig. 8

COR catalyzes the penultimate step in morphine biosynthesis, NADPH-dependent reduction of codeinone to codeine.

Fig. 9. Overlay of CHR (gray) and modeled COR (cyan) active sites

NADP⁺ is shown in gold. Carbon atoms of COR are shown in green; carbon atoms of CHR are shown in gray.