The protein conformational basis of isoflavone biosynthesis

Abstract

Isoflavonoids play important roles in plant defense and also exhibit a range of mammalian health-promoting activities. Their biosynthesis is initiated by two enzymes with unusual catalytic activities; 2-hydroxyisoflavanone synthase (2-HIS), a membrane-bound cytochrome P450 catalyzing a coupled aryl-ring migration and hydroxylation, and 2-hydroxyisoflavanone dehydratase (2-HID), a member of a large carboxylesterase family that paradoxically catalyzes dehydration of 2-hydroxyisoflavanones to isoflavone. Here we report the crystal structures of 2-HIS from Medicago truncatula and 2-HID from Pueraria lobata. The 2-HIS structure reveals a unique cytochrome P450 conformation and heme and substrate binding mode that facilitate the coupled aryl-ring migration and hydroxylation reactions. The 2-HID structure reveals the active site architecture and putative catalytic residues for the dual dehydratase and carboxylesterase activities. Mutagenesis studies revealed key residues involved in substrate binding and specificity. Understanding the structural basis of isoflavone biosynthesis will facilitate the engineering of new bioactive isoflavonoids.

Fig. 1. The enzymatic reactions catalyzed by 2-HIS and 2-HID.

2-HIS catalyzes hydroxylation and aryl-ring migration of flavanones (e.g., liquiritigenin and naringenin) to convert them to 2-hydroxyisoflavanones, and 2-HID then catalyzes dehydration of 2-hydroxyisoflavanones to yield the final isoflavone products, e.g., daidzein or genistein.

Fig. 2. Ribbon diagram of the M. truncatula 2-HIS structure.

The α helices, β strands and the N- and C-termini are labeled. Fig. 1–6 were prepared with MOLSCRIPT and RASTER3D, or PyMOL.

Fig. 3. The heme binding conformation of 2-HIS.

a Heme molecule and its interactions with 2-HIS. b A comparison of the I-helices of 2-HIS (in cyan) and human P450 2C9 (in grey). c,d A comparison of the heme binding loop of 2-HIS (in cyan) and human P450 2C9 (in grey). The structures of heme and key residues involved in heme binding are shown as ball-and-stick models and colored according to element: oxygen, red; nitrogen, blue; sulfur, orange; iron, black for 2-HIS, and grey for P450 2C9; and carbon, green and yellow for 2-HIS residues and heme, and grey and dark grey for P450 2C9 residues and heme. The same atom color codes are used for other figures except ones with specific descriptions. The dashed lines in Fig. 2a indicate the interactions between heme and R433 and T438.

Fig. 4. The substrate-binding pocket and interactions of 2-HIS with docked substrate liquiritigenin.

Substrate liquiritigenin and heme are shown as ball-and-stick models with carbon atoms colored in yellow and iron atom in black. Some amino acid residues in the substrate binding pocket within ~4.5 angstroms from substrate are labeled and shown as ball-and-stick models with carbon atoms colored in green.

Fig. 5. Conformation of the entrance to the 2-HIS active site.

The conformation is shown in two different orientations with the plane of the heme ring perpendicular to the paper and the heme propionate groups pointing to the front (a) and with the heme propionate groups pointing to the right (b). The structure of 2-HIS is illustrated using different colors: the B-C loop (orange), helices F and G and F-G loop (green), helix I and strand β1-4 (cyan); and human P450 2C9 is in grey. The substrate and heme in 2-HIS are shown as thick bond models in yellow and orange, respectively, and the substrate and heme (thin bond model) in human P450 2C9 are in yellow and blue, respectively.

Fig. 6. Proposed 2-HIS reaction mechanism.

a Ring migration: Substrate binds near heme propionate groups (1); heme carboxylate abstracts 3β-hydrogen to deprotonate C3 (2); and B-ring moves from C2 to C3 (3). b Hydroxylation: The dioxygen is bound to the heme iron in the “oxygen binding site”; after the hydroxyl radical is generated, it would be transferred to the “hydroxyl radical transition site” near the heme propionate groups under Ser308, then finally transferred from the activated oxygen intermediate to the C2 atom after the migration of the phenyl B-ring from C2 to C3.

Fig. 7. HPLC profiles of 2-HIS reaction products.

2-HIS enzyme was from dissolved crystals, and substrate liquiritigenin was converted to 2,7,4’-trihydroxyisoflavanone which was subsequently dehydrated to daidzein. The right panels are the HPLC profiles of authentic standards liquiritigenin and daidzein.

Fig. 8. Ribbon diagram of the P. lobata 2-HID structure.

The α helices, β strands and the N- and C-termini are labeled.

Fig. 9. Structural basis for dual functionality of 2-HID from P. lobata.

a 2Fo-Fc electron density map at 1.0 σ for p-nitrophenyl (NPO) and a catalytic triad, Thr168, Asp269 and His301 for carboxylesterase activity; b Active site of 2-HID docked with substrate 2,7,4´-trihydroxyisoflavanone close to the catalytic acid Glu89 and catalytic base His301. Key residues involved in substrate binding are shown as bond models and colored according to element: oxygen, red; nitrogen, blue; and carbon, cyan. NPO and 2,7,4’-trihydroxyisoflavanone are also shown as bond models with their carbon atoms in yellow.