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. 2017 Feb 24;8:248.
doi: 10.3389/fpls.2017.00248. eCollection 2017.

Computational Identification of Amino-Acid Mutations That Further Improve the Activity of a Chalcone-Flavonone Isomerase From Glycine max

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Free PMC article

Computational Identification of Amino-Acid Mutations That Further Improve the Activity of a Chalcone-Flavonone Isomerase From Glycine max

Hui Yuan et al. Front Plant Sci. .
Free PMC article

Abstract

Protein design for improving enzymatic activity remains a challenge in biochemistry, especially to identify target amino-acid sites for mutagenesis and to design beneficial mutations for those sites. Here, we employ a computational approach that combines multiple sequence alignment, positive selection detection, and molecular docking to identify and design beneficial amino-acid mutations that further improve the intramolecular-cyclization activity of a chalcone-flavonone isomerase from Glycine max (GmCHI). By this approach, two GmCHI mutants with higher activities were predicted and verified. The results demonstrate that this approach could determine the beneficial amino-acid mutations for improving the enzymatic activity, and may find more applications in engineering of enzymes.

Keywords: chalcone–flavonone isomerase; enzyme engineering; molecular modeling; positive selection; protein design.

Figures

FIGURE 1
FIGURE 1
The flavonoid pathway. CHS, chalcone synthase; CHR, chalcone reductase; IFS, isoflavone synthase; IOMT, 2′-hydroxyisoflavanone 4′-O-methyltransferase; I2′H, isoflavone 2′-hydroxylase; I3′H, isoflavone 3′-hydroxylase (Liu and Dixon, 2001; Liu et al., 2002, 2003; Shimada et al., 2003).
FIGURE 2
FIGURE 2
Flowchart of the computational approach used in this study.
FIGURE 3
FIGURE 3
The MSA of the CHI core regions. The seven sequences in group 1 are type- II CHIs and another seven sequences in group 2 are type-I CHIs. The sequence positions are numbered according to GmCHI. The subfamily specific positions (SSPs) are marked in green.
FIGURE 4
FIGURE 4
Representative docking poses of the substrate in the active site of GmCHI. (A) The RMSD-binding energy plot for GmCHI. According to the RMSDs with respect to the binding pose of the product, the docking poses could be divided to four groups: I, II, III, and IV. (B–E) The representative poses for groups I, II, III, and IV, respectively (binding pose of the product is shown in gray).
FIGURE 5
FIGURE 5
The RMSD-binding energy plots of 2,000 docking poses of the wild-type enzyme (in blue dots) and the mutants (in red dots). (A) I197P; (B) R110H.

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