Angewandte
Chemie
selective variants are listed in Table S2 in the Supporting
Information.
These initial results demonstrate the viability of our
approach. Some of the identical newly introduced valine
mutations occur in both S,S- and R,R-selective variants,
namely, mutations L114V and L147V. We then tested phenyl-
alanine as the sole building block in a similar manner. In this
case, only 384 transformants (27 ꢀ 3 = 384) were screened for
95% library coverage, because these 10 residues contain
three positions already harboring phenylalanine (F75, F134,
and F139), which need not be included in the randomization.
As summarized in Figure 2B, the best variant L74F/L114F led
to pronounced inversion of enantioselectivity in favor of
(R,R)-2 (e.r. 7:93), whereas the improvement of S,S selectivity
was moderate. In the case of the R,R-selective mutant L74F/
L114F (e.r. 7:93), a cooperative mutational effect[10] is
operating, because variant L114F favors the formation of
(R,R)-2 (e.r. 22:78), whereas L74F is S,S-selective (e.r. 60:40).
To boost enantioselectivity, we applied ISM[1b,2] in two
variations, again keeping minimal screening in mind. In one
case, the gene of variant SZ19 (L103V/L114V/I116V/F139V/
L147V) was used as a template for randomization at residues
L74, M78, and I80 by employing phenylalanine as the sole
building block. The three amino acid positions do not occur in
the template SZ19, and the remaining residues F75 and F134
already feature phenylalanine. In this way, we identified
variants SZ92 and SZ95 with high S,S selectivity (e.r. 96:4;
Figure 2C). In another experiment, mutant SZ118 (L74F/
L114F) was chosen as the template for improving R,R
selectivity. This time randomization was performed with
valine as the building block and resulted in the generation of
three highly enantioselective variants (Figure 2D), the best of
which was SZ338 (L74F/L114F/M78V/I80V) with e.r. 2:98.
Although numerous enantioselective enzyme mutants
have been generated in previous directed-evolution studies,
X-ray structural analyses were hardly ever performed.[2c,11] To
identify structural differences between WT LEH and the best
R,R- and S,S-selective mutants, we solved the X-ray crystal
structure of SZ338 at 2.05 ꢁ and that of SZ92 at 1.53 ꢁ. When
the structures of SZ92 and SZ338 were superimposed on that
of WT LEH (PDB ID: 1NWW), only very small root-mean-
square deviations of 0.254 and 0.424 ꢁ, respectively, were
observed for the backbone Ca atoms (Figure 3A). The most
obvious differences between the three structures are located
in the flexible loops and the C-terminal region. As compared
to those in the WT and SZ92 mutant structures, the C-
terminal a helix (residues 138–142) and the loop between
residues 55 and 60 in the SZ338 structure have shifted by
about 7.2 and 4.3 ꢁ, respectively. These conformational
changes may be the consequence of the L114F mutation.
We also determined the product-containing (complexed)
structures of mutant SZ92 harboring (S,S)-2 at 1.70 ꢁ and
mutant SZ338 with (R,R)-2 at 2.25 ꢁ by soaking the
respective crystals with epoxide 1 (Figure 3B,C). As shown
in Figure 3E,F, the binding pocket in the complexed structure
of SZ338 with (R,R)-2 has been significantly downsized and
closed. This structural change may be caused by motion of the
C-terminal helix and a further side-chain shift of F74.
Although both SZ92 and SZ338 harbor the L74F mutation,
Figure 3. Structure of LEH variants. A) Superposition of the LEH wild-
type (light blue), SZ92 (pale yellow), and SZ338 (light pink) structures.
The helix 136–142 and loop 55–60 of SZ338 are shifted by 7.2 and
4.3 ꢂ, respectively. B,C) Structures of SZ92 complexed with (S,S)-
cyclohexanediol (B) and SZ338 complexes with (R,R)-cyclohexanediol
(C). The catalytic residues D101/D132 and the mutated residues are
shown as gray balls and sticks. The 2jFo jꢀj2Fc j electron-density map
of ligands (blue mesh) are contoured at 1.0 s. D) Comparison of the
F74 rotamer conformation in LEH variants. Light blue and orange
represent apo and complexed wild-type LEH, respectively; pale yellow
and marine are apo and complexed SZ92, respectively; light pink and
cyan are apo and complexed SZ338, respectively. E,F) Surface repre-
sentation of the active pocket in apo (E) and complexed (F) structures.
The catalytic residue D101 is shown in gray as a ball-and-stick model.
only the SZ338 complexed structure contains a dual con-
formation of the F74 side chain (Figure 3D). The occupancy
ratio of rotamer 1 and rotamer 2 has been shifted from 6.5:1.5
in the apo structure of SZ338 to 1:6 in the SZ338 complexed
structure. However, only rotamer 1 of F74 occurs in the apo
and complexed structures of SZ92. Interestingly, there are
also two rotamer conformations in the crystal structures of
WT LEH, with rotamer 1 found only in the apo structure
(PDB ID: 1NWW), whereas rotamer 2 is found only in the
complexed structure (PDB ID: 1NU3). The detailed con-
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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