Fig 1.
Antioxidant flavonoids produced by DaCHI1 in response to various types of oxidative stresses.
Substrate structures and specificities of type I and II CHIs are shown. CHS, chalcone synthase; PKR, NADPH-dependent chalcone reductase.
Table 1.
Primer used in this study.
Table 2.
Data collection and refinement statistics.
Fig 2.
DaCHI1 transcription under cold and UV irradiation treatments.
Plants grown at 16°C and 30 μmol m−2 s−1 light were subjected to cold and UV-B stress. Plants were exposed to a temperature of 0°C for 2 days or UV-B irradiation (2 kJ m−2 day−1) for 1 day after 1-day dark adaptation. RNAs extracted from leaves of stress-treated plants were used for RT-PCR of the DaCHI1 gene, with 18S rRNA as an internal control.
Fig 3.
Crystal structure of DaCHI1 and multiple sequence alignment of CHI proteins.
(A) Overall structure of unliganded DaCHI1, shown as a ribbon diagram. α-Helices and β-strands are colored cyan and orange, respectively. (B) Overall structure of isoliquiritigenin-complexed DaCHI1, shown as a ribbon diagram. The bound ligand is shown as a stick model with a Fo-Fc electron density map (contoured at 3σ). (C) Multiple sequence alignment of DaCHI1, ZmCHI (Zea mays; UniProtKB code Q08704), AtCHI (PDB code 4DOI; UniProtKB code P41088), VvCHI (Vitis vinifera; NCBI reference sequence NP_001268033.1), PhCHI (Petunia hybrida; UniProtKB code P11651), IpCHI (Ipomoea purpurea; UniProtKB code O22604), MsCHI (Medicago sativa; UniProtKB code P28012), PvCHI (Phaseolus vulgaris; NCBI reference sequence XP_007142690.1), and PsCHI (Pisum sativum; UniProtKB code P41089). Strictly and partially conserved residues are shaded black and gray, respectively. Two residues for distinguishing between type I and II CHI are indicated by black circles above the sequence alignment. Secondary structures obtained from the crystal structure of DaCHI1 are shown above the aligned sequence. Multiple sequence alignment was performed with ClustalX and was edited with the GeneDoc program.
Table 3.
Structural homologs of DaCHI1 selected from a DALI search (DALI-lite server).
Fig 4.
(A) Empty ligand-binding site and (B) active site residues of the unliganded DaCHI1 structure. The isoliquiritigenin binding mode of DaCHI1 is shown. The ligand is depicted as a stick figure with green carbon atoms; interacting residues of DaCHI1 are shown as bright orange sticks. Hydrogen bonds with bound isoliquiritigenin are indicated by a red dotted line. (C) Structural superposition of the ligand-binding regions of unliganded DaCHI1 (cyan) and isoliquiritigenin (green)-bound DaCHI1 (bright orange) showing the closing of the β3–β4 region by ligand binding. Hydrophobic interactions are indicated by yellow dotted lines.
Table 4.
Steady-state kinetic parameters of wild-type and mutant DaCHI1.
Fig 5.
Structural comparison between isoliquiritigenin-bound DaCHI1 and liquiritigenin-bound MsCHI.
(A) Isoliquiritigenin (orange)-bound DaCHI1 and liquiritigenin (yellow)-bound MsCHI structures are shown. (B) Liquiritigenin-binding mode of MsCHI. (C) Superposition of isoliquiritigenin (orange)-bound DaCHI1 (dark gray) and liquiritigenin (yellow)-bound MsCHI (light gray) structures. Overall ligand-binding modes differed markedly between the two complexes; the liquiritigenin-binding position was shifted deep into the active site cavity in MsCHI as compared to isoliquiritigenin binding in DaCHI1. Conformational differences between the two structures were also observed at the Arg34 position (Arg36 in MsCHI) and in the β6–β7 loop region.
Fig 6.
Activity and stability of DaCHI1.
(A) Effects of temperature on DaCHI1 activity. Enzymatic activity was evaluated in the temperature range of 0–70°C in 50 mM potassium phosphate buffer (pH 7.6). Activity is expressed as a percentage of the maximum activity (100%). (B) Effects of temperature on the stability of DaCHI1. The enzyme was pre-incubated for 30 min at temperatures ranging from 0–60°C. Residual activity was measured at 25°C. (C) pH dependence of DaCHI1 activity. The reaction was carried out at 25°C in buffers with pH ranging from 3.0 to 10.0. The following buffers were used: pH 3.0–6.0, 50 mM citrate; pH 6.0–8.0, 50 mM potassium phosphate; and pH 8.0–10.0, 50 mM Tris-HCl. All measurements were made in triplicate.