Enzyme Toolbox: Novel Enantiocomplementary Imine Reductases

Article abstract of DOI:10.1002/cbic.201402213

Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O

Full text of DOI:10.1002/cbic.201402213

CHEMBIOCHEM
COMMUNICATIONS
DOI: 10.1002/cbic.201402213
Enzyme Toolbox: Novel Enantiocomplementary Imine
Reductases
[
a]
[a]
[a]
[a]
Philipp N. Scheller, Silvia Fademrecht, Sebastian Hofelzer, Jꢀrgen Pleiss,
[b]
[b]
[a]
[a]
Friedemann Leipold, Nicholas J. Turner, Bettina M. Nestl,* and Bernhard Hauer*
Reducing reactions are among the most useful transformations
for the generation of chiral compounds in the fine-chemical
industry. Because of their exquisite selectivities, enzymatic ap-
proaches have emerged as the method of choice for the re-
duction of C=O and activated C=C bonds. However, stereo-
selective enzymatic reduction of C=N bonds is still in its infan-
cy—it was only recently described after the discovery of en-
zymes capable of imine reduction. In our work, we increased
the spectrum of imine-reducing enzymes by database analysis.
By combining the currently available knowledge about the
function of imine reductases with the experimentally uncharac-
terized diversity stored in protein sequence databases, three
novel imine reductases with complementary enantiopreference
were identified along with amino acids important for catalysis.
Furthermore, their reducing capability was demonstrated by
the reduction of the pharmaceutically relevant prochiral imine
recently introduced organocatalytic methods. Although much
progress was made in terms of selectivity, sustainable prepara-
tion remains challenging because of the numerous deprotec-
tion steps and the application of expensive and toxic
[2,4]
metals.
Enzymatic approaches using w-transaminases, lipas-
es, amine oxidases, and amine dehydrogenases provide alter-
natives to the classical chemical methods, as the high specifici-
ty of enzymes routinely allows the preparation of chiral inter-
[
3,5–9]
mediates in pure forms.
Biocatalytic methods for the re-
[10]
duction of activated C=C bonds by ene reductases and C=O
bonds by ketoreductases are well described. The impressive
progress in recent years has made ketoreductases the first
[11]
choice for ketone reductions, which are as a consequence
[5,12,13]
applied in several processes on an industrial scale.
The asymmetric reduction of prochiral C=N bonds for the
preparation of chiral amines is a well-established route in syn-
thetic chemistry because of the easy accessibility of imines
from their ketone precursors, with the asymmetric addition of
2-methylpyrroline. These novel enzymes exhibited comparable
to higher catalytic efficiencies than previously described en-
zymes, and their biosynthetic potential is highlighted by the
full conversion of 2-methylpyrroline in whole cells with excel-
lent selectivities.
[2]
hydrogen or a hydride as the key stereo-differentiating step.
Until recently, the biocatalytic counterparts were rarely de-
scribed in the literature and restricted to some strains that are
able to reduce imine bonds to amines in a stereospecific
[14–17]
manner.
This was explained by the unstable C=N bond in
Enantiopure chiral amines are core components of a large
number of pharmaceuticals, agrochemicals, and other specialty
aqueous environments and its rapid hydrolysis to the aldehyde
[14,17]
or ketone precursor.
Enzymatic asymmetric reduction of
[
1]
chemicals. Their enormous variety of biological activities is
not surprising given their high structural diversity and propen-
sity for hydrogen bond formation. Despite the historical need
for and continued interest in chiral amines, there is still a lack
of generally applicable synthetic methods for amine produc-
tion, and the classical resolution of racemic materials remains
imines by imine reductases (IREDs) was therefore mainly re-
stricted to the few naturally occurring imines, like dihydrofo-
1
[18,19]
late and D -pyrroline-2-carboxylate.
This changed recently
after Nagasawa and co-workers identified, purified, and charac-
terized three enantiocomplementary IREDs from different
[20–22]
Streptomyces strains:
two R- and one S-specific reductase
[
2,3]
the method of choice on industrial scales.
Asymmetric syn-
((R)-IRED and (S)-IRED), were reported.
thesis by reducing reactions is the most frequently applied ap-
proach for the generation of chiral molecules, and the main
chemical strategies for enantioselective reductions involve
transition-metal-mediated homogenous catalysis as well as
These enzymes were able to reduce the prochiral 2-methyl-
pyrroline 1 to the pharmaceutically relevant five-membered
endocyclic (R)-2-methylpyrrolidine ((R)-2) and (S)-2-methylpyr-
[20–22]
rolidine ((S)-2)
(Scheme 1); with (R)-2 as an important
[23,24]
building block for new histamine H receptor antagonists
3
[a] P. N. Scheller, S. Fademrecht, S. Hofelzer, Prof. Dr. J. Pleiss, Dr. B. M. Nestl,
Prof. Dr. B. Hauer
Institute of Technical Biochemistry, University of Stuttgart
Allmandring 31, 70569 Stuttgart (Germany)
E-mail: bettina.nestl@itb.uni-stuttgart.de
bernhard.hauer@itb.uni-stuttgart.de
[b] Dr. F. Leipold, Prof. Dr. N. J. Turner
Manchester Institute of Biotechnology, University of Manchester
1
31 Princess Street, M1 7DN Manchester (UK)
Scheme 1. Asymmetric reduction of 2-methylpyrroline 1 to enantiopure 2-
methylpyrrolidines (R)-2 or (S)-2 by enantiocomplementary imine reductases.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402213.
ꢁ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 4
&
1
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
[
25]
and HIV reverse transcriptase inhibitors.
These enzymes
Imine Reductase Engineering Database).
might offer an alternative route to (R)-2, as the shortest enan-
Each of these genes was synthesized in codon-optimized
form for expression in Escherichia coli with an N-terminal His6
tag to facilitate purification. Biocatalyst production was per-
formed by using the arabinose-inducible pBAD expression
tioselective synthesis comprises a time-consuming four step
[
26]
procedure.
Shortly after the discovery of the novel IREDs, the molecular
[29]
structure of the R-selective IRED from Streptomyces kanamyceti-
system in E. coli strain JW5510,
and overnight expression
[
27]
cus ((R)-IRED-Sk) was solved by X-ray crystallography, and the
biosynthetic potential of (S)-IRED from Streptomyces sp.
GF3546 ((S)-IRED-Ss) was explored with a variety of cyclic
yielded soluble, active enzymes. Initial experiments with cell
lysate indicated NADPH-dependent reductase activity with 1 as
substrate. In addition, NADH was tested as a cofactor, but no
enzymatic activity was detected. In controls (lysate of cells
with an empty vector) no increase in NADPH oxidation rate
was detected after the addition of 1.
[
28]
imines. However, the promising imine reductase superfamily
consists so far of only a few described members.
To expand the scope of known IREDs, we established the
Imine Reductase Engineering Database (http://www.ired.uni-
stuttgart.de) with more than 350 protein entries. By sequence
similarity to the described IREDs, two novel R-type and one S-
type IRED were selected from our database for confirmation of
their catalytic activity. Here, we report the production and ini-
tial characterization of these enzymes and their use as whole-
cell biocatalysts for the reduction of 1. The novel imine reduc-
tases showed excellent enantioselectivities. The enantioprefer-
ences of the two R-type and the S-type IREDs were deter-
mined, thus confirming their database assignment in R-IRED-
type and S-IRED-type superfamilies. Moreover, functionally rele-
vant amino acids were identified by conservation analysis and
verified by site-directed mutagenesis.
For activity assays and biotransformations, the enzymes
2
+
were purified to homogeneity by Ni -affinity chromatography.
Purified IREDs have been described to function as di-
[21,22,27]
mers,
and SDS-PAGE analysis confirmed that the purified
enzymes had the expected molecular weight (32–36 kDa per
monomer; Figure S3).
To evaluate imine reduction activity with purified enzymes,
in vitro biotransformations were performed for 24 h, with
10 mm 1 as substrate and glucose-6-phosphate/glucose-6-
phosphate dehydrogenase as a cofactor regeneration system.
For (R)-IRED-Sr excellent substrate conversion was observed:
9.74 mm product was detected after 0.5 h. High activities were
also obtained with (R)-IRED-St: 9.91 mm 2 detected after 1 h.
Similar conversion rates (~95%) were observed with the enan-
tiocomplementary (S)-IRED-Pe within 24 h (Tables S6–S8 and
Figures S4–S6).
For the establishment of the Imine Reductase Engineering Da-
tabase a BLAST search was performed with the characterized
[
27]
[22]
IREDs, (R)-IRED-Sk and (S)-IRED-Ss. Sequences with E values
À30
below 10 were collected and assigned to families according
To avoid the use of the expensive and unstable NADPH co-
factor and its regeneration system, the performance of cells ex-
pressing these enzymes as whole-cell biocatalysts was investi-
to sequence similarity. Sequence similarity network analyses
revealed a clear separation into two superfamilies (Figure S1 in
the Supporting Information): the smaller superfamily (74 pro-
teins) includes (S)-IRED-Ss, and the larger superfamily (282 pro-
teins) includes the two previously characterized (R)-IREDs.
To select new IREDs, a conservation analysis was performed
for each superfamily (Table S2). Promising candidates were
chosen based on high sequence similarity to the characterized
À1
gated (final OD600 =30 in biotransformations, ꢀ50 mgmL cell
wet weight). Table 1 shows the data of 2-methylpyrroline 1 con-
version with wild-type and over 24 h, with particular focus on
the initial time points 0.5 and 1 h. Under optimized conditions
with resting E. coli cells and glucose for in situ cofactor regen-
eration, full substrate conversion was achieved with all strains
expressing wild-type enzymes. For cells expressing (R)-IRED-Sr
and (R)-IRED-St, a 3 h reaction time was sufficient for the com-
plete conversion of substrate 1 (see also Table S9). As previous-
ly observed in in vitro biotransformations, an increased reac-
tion time (8 h) was required for (S)-IRED-Pe to obtain complete
conversion (Tables 1 and S9). In contrast, cells harboring an
[
27]
[20]
IREDs (R)-IRED-Sk and (R)-IRED-Ss as well as to (S)-IRED-
[
22]
Ss, provided they had no significant deviation at the con-
served positions (Tables S3–S5 and Figure S2). The selected R-
type IRED and S-type IRED sequences were derived from Strep-
tosporangium roseum DSM 43021 ((R)-IRED-Sr, 60.8% sequence
identity to (R)-IRED-Sk), Streptomyces turgidiscabies ((R)-IRED-St,
80.7% sequence identity to (R)-IRED-Ss), and Paenibacillus elgii
((S)-IRED-Pe, 60.5% sequence
identity to (S)-IRED-Ss). Detailed
information on the selected
IREDs is given in the Supporting
Information. (R)-IRED-Sr is anno-
Table 1. Product formations [mm] and enantiomeric excesses of 2-methylpyrrolidine 2 from biotransformations
with imine reductases wild-type and mutant whole cells.
[a]
IRED variant
Reaction time [h]
8
ee [%]
+
tated as an NAD -dependent
0.5
1
24
glycerol-3-phosphate dehydro-
genase in GenBank, whereas
(
R)-IRED-Sr
4.63Æ0.04
2.96Æ0.15
1.87Æ0.01
0.72Æ0.01
0.11Æ0.02
–
8.98Æ0.29
6.46Æ0.42
3.81Æ0.04
1.49Æ0.03
0.22Æ0.01
–
10.75Æ0.93
10.69Æ0.57
9.45Æ0.13
5.83Æ0.42
1.34Æ0.03
<0.05
10.32Æ0.63
98.3 R
99.0 R
94.9 S
91.4 R
94.1 R
65.7 S
(R)-IRED-St
10.38Æ0.47
9.68Æ0.10
6.37Æ0.38
1.74Æ0.24
<0.1
(
(
(
(
S)-IRED-Pe
(
R)-IRED-St and (S)-IRED-Pe are
R)-IRED-Sr D170A
R)-IRED-St D172A
S)-IRED-Pe Y187A
classified as 6-phosphogluco-
nate dehydrogenases, which are
the most common GenBank an-
notations for members of the
[
a] Enantiomeric excesses of the products were determined after 24 h; error bars were in general below 0.1%.
ꢁ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 4
&
2
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
empty pBAD33 plasmid (negative control) showed no conver-
sion.
all IREDs, only five differ between the two superfamilies
(Figure 1 purple residues, Table S2). All are located at a distance
of only 9 ꢂ from the nicotinamide ring of the co-crystallized
NADPH in the (R)-IRED-Sk crystal structure (Uniprot: Q1EQE0,
PDB: 3ZHB). Four of these residues (Leu34, Cys82, Thr110, and
Ile136 in (R)-IRED-Sk; Met36, Ser84, Ser111, and Val137 in (S)-
IRED-Pe) are in the N-terminal half of the enzyme with the
Rossman fold and its GXGXXG motif (typically associated with
NADPH binding). The most striking difference between the su-
perfamilies was at position 187: aspartic acid in (R)-IRED-Sk but
tyrosine in (S)-IRED-Pe.
Furthermore, the selectivity of the in vivo imine bioreduc-
tions was determined by chiral gas chromatography with
flame ionization detection (GC-FID) analysis. (R)-IRED-Sr and
(
R)-IRED-St formed the R isomer of 2 with excellent enantio-
meric excess (98.3 and 99.0%, respectively). (S)-2 was produced
with 94.9% ee by cells expressing (S)-IRED-Pe (Table 1, Fig-
ure S7).
To explore these differences in conversion rates at the mo-
lecular level and to be able to compare our newly discovered
enzymes with already characterized IREDs, kinetic parameters
were determined by using a NADPH-based spectrophotometric
assay.
Both residues are located in the interdomain helix and are
highly conserved in their respective superfamilies (86% Asp
and 11% Glu in the (R)-IRED-type superfamily; 97% Tyr in the
(S)-IRED superfamily). Mutational analysis of Asp187 in (R)-
IRED-Sk was reported by Rodrꢃguez-Mata et al.: substitution by
In agreement with the biotransformation results, the catalyt-
ic efficiency of (R)-IRED-Sr was much higher than for (R)-IRED-St
[27]
Asn or Ala resulted in an inactive enzyme. Hence, they pro-
posed a mechanism for imine reduction initiated by a catalytic
Asp protonating the imine to an iminium ion; the iminium ion
is stabilized by negatively charged amino acids in the active
site and subsequently reduced by a hydride transfer from
Table 2. Kinetic parameters of purified IREDs with 1 as substrate.
À1
À1
À1
Enzyme
K
M
[mm]
kcat [min
]
M i
kcat/K [min mm ] K [mm]
(R)-IRED-Sr 1.08Æ0.13 57.63Æ1.44 53.13Æ6.58
(R)-IRED-St 1.81Æ0.07 26.73Æ1.36 14.73Æ0.93
(S)-IRED-Pe 1.29Æ0.15 1.58Æ0.09 1.22Æ0.16
28.40Æ3.01
21.07Æ1.48
33.28Æ8.02
[27]
NADPH.
We observed that mutation of this Asp to an Ala in (R)-IRED-
Sr and (R)-IRED-St resulted in decreased conversion rates (to 15
[30]
and 5%, respectively (initial time points ) Table 1) in whole
cell biotransformations, but not in complete inactivation as
previously described for (R)-IRED-Sk. The residual activity indi-
cates the presence of other proton-donating amino acids in
the large active site of (R)-IREDs, or the coordinating function
of Asp187 with water as a proton donor.
and (S)-IRED-Pe. K values were between 1.1 and 1.8 mm, how-
ever kcat varied over a wider range (1.5 to ~57 min ; Table 2).
Hence, the catalytic efficiencies of the novel R-specific IREDs
M
À1
(
(R)-IRED-Sr and (R)-IRED-St) are over two orders of magnitude
[
27]
higher than that reported for (R)-IRED-Sk. In contrast, the
kinetic parameters for (S)-IRED-Pe were slightly higher than for
We hypothesized that in the S-IRED-type superfamily the
conserved Tyr (equivalent position to Asp187) might also serve
as the proton-donating amino acid. Indeed, mutation of Tyr187
to an Ala rendered the enzyme nearly completely inactive (<
0.1 mm product after 24 h and loss of selectivity; Table 1).
In summary, we have demonstrated that previously un-
known and uncharacterized imine reductases are able to ste-
[
28]
(
S)-IRED-Ss; this results in about the same range of catalytic
efficiency. Substrate inhibition has been reported for
[
27,28]
IREDs,
and was also observed here: (R)-IRED-Sr, (R)-IRED-St,
and (S)-IRED-Pe showed decreased activities at higher concen-
trations (Tables S10–S12 and Figures S8–S10).
The relatively low catalytic efficiencies of IREDs imply that
1
is not the natural substrate of
these enzymes (as also indicat-
ed by their GenBank annota-
tions). To exclude dehydrogen-
ase activity of IREDs with glycer-
ol-3-phosphate and 6-phospho-
gluconate as substrates, their
oxidation rate was monitored
with purified enzymes. Based
+
+
on NADP and NAD reduction
rates, no activity was detected.
For a better understanding of
the imine-reducing activity of
these enzymes, potential cata-
lytically relevant amino acids
were identified by systematical-
ly comparing the conserved
amino acid positions of R-type
IREDs and S-type IREDs. Of the
Figure 1. Schematic active site of a R-type IRED (left) based on (R)-IRED-Sk crystal structure, and of an S-type IRED
(right) based on a homology model of (S)-IRED-Pe. Imine reductase backbones are shown (ribbons) with subunits
A (green) and B (blue) and NADPH (black sticks). Amino acids (Leu34, Cys82, Thr110, Ile136, and Asp187 in (R)-
IRED-Sk; Met36, Ser84, Ser111, Val137, and Tyr187 in (S)-IRED-Pe) being differentially conserved among the two
IRED superfamilies’ are highlighted as purple sticks.
53 positions conserved across
ꢁ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 4
&
3
&
ÞÞ
These are not the final page numbers!

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
reoselectively reduce endocyclic 2-methylpyrroline to the cor-
responding amine product. A database analysis identified nu-
merous genes with unknown functions that belong to mem-
bers of two IRED superfamilies; R- and S-selective enzymes
were demonstrated as three novel examples. We further
showed that these newly discovered enzymes exhibit higher
[6] M. Hall, A. S. Bommarius, Chem. Rev. 2011, 111, 4088–4110.
[
7] M. J. Abrahamson, E. Vꢄzquez-Figueroa, N. B. Woodall, J. C. Moore, A. S.
Bommarius, Angew. Chem. Int. Ed. 2012, 51, 3969–3972; Angew. Chem.
2
012, 124, 4036–4040.
[8] H. Li, J. C. Liao, ACS Synth. Biol. 2014, 3, 13–20.
[9] B. M. Nestl, S. C. Hammer, B. A. Nebel, B. Hauer, Angew. Chem. Int. Ed.
2
014, 53, 3070–3095; Angew. Chem. 2014, 126, 3132–3158.
10] R. Stuermer, B. Hauer, M. Hall, K. Faber, Curr. Opin. Chem. Biol. 2007, 11,
03–213.
[
(one to two orders of magnitude) catalytic activities for 2-
2
methylpyrroline than reported for previously described IREDs.
The potential of our new enzymes for biosynthetic purposes
was highlighted in the asymmetric biotransformations that led
to complete substrate conversion in short reactions times, by
using purified enzymes and whole cells. (R)- and (S)-2-methyl-
pyrrolidine products were obtained in up to 99% enantiomeric
excess. Finally, we identified residues involved in catalysis by
comparing R-type and S-type IREDs. The significantly de-
creased conversion rates of these mutants indicate that the
targeted amino acid residues are crucial for the reducing activi-
ty. This might help to achieve a better understanding of imine
reduction by these enzymes and provide a basis for enzyme
engineering approaches.
[11] J. C. Moore, D. J. Pollard, B. Kosjek, P. N. Devine, Acc. Chem. Res. 2007,
40, 1412–1419.
[
12] U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K.
Robins, Nature 2012, 485, 185–194.
[
13] S. M. A. De Wildeman, T. Sonke, H. E. Schoemaker, O. May, Acc. Chem.
Res. 2007, 40, 1260–1266.
[14] H. Li, P. Williams, J. Micklefield, J. M. Gardiner, G. Stephens, Tetrahedron
004, 60, 753–758.
2
[
[
15] T. Vaijayanthi, A. Chadha, Tetrahedron: Asymmetry 2008, 19, 93–96.
16] M. Espinoza-Moraga, T. Petta, M. Vasquez-Vasquez, V. F. Laurie, L. A. B.
Moraes, L. S. Santos, Tetrahedron: Asymmetry 2010, 21, 1988–1992.
[17] K. Mitsukura, M. Suzuki, K. Tada, T. Yoshida, T. Nagasawa, Org. Biomol.
Chem. 2010, 8, 4533–4535.
[
18] M. Whitlow, A. J. Howard, D. Stewart, K. D. Hardman, L. F. Kuyper, D. P.
Baccanari, M. E. Fling, R. L. Tansik, J. Biol. Chem. 1997, 272, 30289–
30298.
[
19] M. Goto, H. Muramatsu, H. Mihara, T. Kurihara, N. Esaki, R. Omi, I. Miya-
hara, K. Hirotsu, J. Biol. Chem. 2005, 280, 40875–40884.
Acknowledgements
[20] T. Nagasawa, T. Yoshida, K. Ishida, H. Yamamoto, N. Kimoto, (Daicel
Chemical Industries, Ltd.), US 20110287494, 2011.
[
[
[
[
21] K. Mitsukura, M. Suzuki, S. Shinoda, T. Kuramoto, T. Yoshida, T. Nagasa-
wa, Biosci. Biotechnol. Biochem. 2011, 75, 1778–1782.
22] K. Mitsukura, T. Kuramoto, T. Yoshida, N. Kimoto, H. Yamamoto, T. Naga-
sawa, Appl. Microbiol. Biotechnol. 2013, 97, 8079–8086.
23] M. Cowart, J. K. Pratt, A. O. Stewart, Y. L. Bennani, T. A. Esbenshade, A. A.
Hancock, Bioorg. Med. Chem. Lett. 2004, 14, 689–693.
24] M. Sun, C. Zhao, G. A. Gfesser, C. Thiffault, T. R. Miller, K. Marsh, J.
Wetter, M. Curtis, R. Faghih, T. A. Esbenshade, A. A. Hancock, M. Cowart,
J. Med. Chem. 2005, 48, 6482–6490.
The research leading to these results has received support from
the Innovative Medicines Initiative Joint Undertaking under grant
agreement no. (115360) resources that are composed of financial
contributions from the European Union’s Seventh Framework
Programme (FP7/2007-2013) and EFPIA companies’ in kind con-
tribution. We would also like to acknowledge Constantin Vogel
and Waldemar Reusch for providing the database system and for
their work on the web interface.
[
[
[
[
[
[
25] K. Krajewski, Y. Zhang, D. Parrish, J. Deschamps, P. P. Roller, V. K. Pathak,
Bioorg. Med. Chem. Lett. 2006, 16, 3034–3038.
26] D. Zhao, J. T. Kuethe, M. Journet, Z. Peng, G. R. Humphrey, J. Org. Chem.
2
006, 71, 4336–4338.
Keywords: asymmetric catalysis
· biocatalysis · database
27] M. Rodrꢃguez-Mata, A. Frank, E. Wells, F. Leipold, N. J. Turner, S. Hart,
J. P. Turkenburg, G. Grogan, ChemBioChem 2013, 14, 1372–1379.
28] F. Leipold, S. Hussain, D. Ghislieri, N. J. Turner, ChemCatChem 2013, 5,
analysis · enantiopure amine · imine reductase
[1] M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Kesseler, R. Stꢀrmer, T. Ze-
3505–3508.
linski, Angew. Chem. Int. Ed. 2004, 43, 788–824; Angew. Chem. 2004,
29] T. Baba, T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsen-
ko, M. Tomita, B. L. Wanner, H. Mori, Mol. Syst. Biol. 2006, 2, 2006.0008.
30] The comparison was performed with initial conversion rates because of
full substrate conversion after 3 h with wild-type enzymes.
116, 806–843.
[
[
[
2] T. C. Nugent, M. El-Shazly, Adv. Synth. Catal. 2010, 352, 753–819.
3] D. Ghislieri, N. J. Turner, Top. Catal. 2014, 57, 284–300.
4] Y. S. Gee, K. F. Abd El Kader, C. J. T. Hyland, Annu. Reports Sect. B 2013,
1
09, 103.
5] F. Hollmann, I. W. C. E. Arends, D. Holtmann, Green Chem. 2011, 13,
285–2314.
[
Received: May 5, 2014
Published online on && &&, 0000
2
ꢁ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 4
&
4
&
ÞÞ
These are not the final page numbers!

COMMUNICATIONS
Let’s transfer hydrides to imines. Bio-
catalytic preparation of chiral amines by
imine reductases is an emerging tech-
nology but is limited to few described
enzymes. Several hundred additional
enantiocomplementary imine reductas-
es sequences were collected in a data-
base, and the biochemical potentials of
three of them are reported. Site-direct-
ed mutagenesis revealed key amino
acid residues for catalysis and stereo-
selectivity.
P. N. Scheller, S. Fademrecht, S. Hofelzer,
J. Pleiss, F. Leipold, N. J. Turner,
B. M. Nestl,* B. Hauer*
&& – &&
Enzyme Toolbox: Novel
Enantiocomplementary Imine
Reductases
ꢁ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 4
&
5
&
ÞÞ
These are not the final page numbers!