Figure 1.
Reduction of 2-chloro-1-phenylethanone to two enantiomeric alcohols by WT-DKR and mutant W222F.
WT-DKR display Re face preference for production of R-alcohol, whereas W222F favors the Si face of the ketone.
Figure 2.
Crystal structure and substrate binding modes of the WT-DKR-NAD+ complex.
(A) α-helices 11, 13 and 14, and β-strands 7, 8 in subunit A, and α-helix 12 in subunit B form the hydrophobic pocket. Hydrophobic residues located at the active site are shown as sticks with the same ribbon color. Trp222 is shown as a sphere in magenta. NAD is shown as a stick in red. (B) Electrostatic interaction between residues located at the α-helix 12 terminus.
Table 1.
Primers used for mutagenesis.
Table 2.
Comparison of enantiopreference toward 2-chloro-1-phenylethanone for WT-DKR and nine DKR mutants.
Figure 3.
UAAs used for Trp222 substitution.
Table 3.
MALDI-TOF/MS analyses of mutants containing unnatural amino acids (UAAs) after tryptic digestion.
Table 4.
Comparison of kinetic parameters and binding affinity of WT-DKR and nine mutantsa,b.
Figure 4.
Helical conformation disruption by mutations at residue 222.
Residues located at the α-helix 12 terminus are displayed as light gray sticks. Mutated residues are highlighted in magenta.
Figure 5.
Comparison of the hydrophobic pocket for the nine DKR mutants and WT-DKR.
In mutants containing V, L, M, F, Y and CNF at position 222, the α-helix 14 in DKR subunit A was pulled away from α-helix 12 in subunit B, and α-helix 11 in subunit A yielded a wider entrance (A, B, C, D, E, F). For MeOF, BiF, and BuOF, α-helix 14 in subunit A was closer to α-helix 11, consequently forming a tighter entrance for the substrate (G, H, I). Amino acids at residue 222 in subunit B are shown as magenta sticks. WT-DKR and mutants are colored light gray and deep teal, respectively.
Table 5.
Docking score and binding free energy of 2-chloro-1-phenylethanone with different DKR mutantsa.
Figure 6.
Substrate binding modes in the active center of WT and mutant DKR.
For Si-preference enzymes (A, B, C, D, E, F), substrate orientations to produce different enantiomers randomly exist in the binding pocket. For Re-preference enzymes (G, H, I, J), a substrate that yields an S-enantiomer (yellow) adopts a “phenyl group first” position formed a pro-(S)-configuration, while the substrate (cyan) prefers the mode of “chloride first” and a pro-(R)-configuration. The substrate orientated in the pro-(S)-configuration is colored gold, and the substrate orientated in the pro-(R)-configuration is cyan. (A) W222V; (B) W222L; (C) W222M; (D) W222F; (E) W222Y; (F) W222CNF; (G) WT-DKR; (H) W222MeOF; (I) W222BiF; (J) W222BuOF.