Stereoselectivity and Structural Characterization of an Imine Reductase (IRED) from Amycolatopsis orientalis

Article abstract of DOI:10.1021/acscatal.6b00782

The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4) catalyzes the NADPH-dependent reduction of a wide range of prochiral imines and iminium ions, predominantly with (S)-selectivity and with ee's of up to >99%. AoIRED displays up to 100-fold greater catalytic efficiency for 2-methyl-1-pyrroline (2MPN) compared to other IREDs, such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)-selectivity, and thus, AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic properties, with inversion of stereoselectivity observed between structurally similar substrates, and also, in the case of 1-methyl-3,4-dihydroisoquinoline, for the same substrate, dependent on the age of the enzyme after purification. The structure of AoIRED has been determined in an "open" apo-form, revealing a canonical dimeric IRED fold in which the active site is formed between the N- and C-terminal domains of participating monomers. Co-crystallization with NADPH gave a "closed" form in complex with the cofactor, in which a relative closure of domains, and associated loop movements, has resulted in a much smaller active site. A ternary complex was also obtained by cocrystallization with NADPH and 1-methyl-1,2,3,4-tetrahydroisoquinoline [(MTQ], and it reveals a binding site for the (R)-amine product, which places the chiral carbon within 4 ? of the putative location of the C4 atom of NADPH that delivers hydride to the C? -N bond of the substrate. The ternary complex has permitted structure-informed mutation of the active site, resulting in mutants including Y179A, Y179F, and N241A, of altered activity and stereoselectivity.

Full text of DOI:10.1021/acscatal.6b00782

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Article
Stereoselectivity and Structural Characterization of an
Imine Reductase (IRED) from Amycolatopsis orientalis
Godwin A. Aleku, Henry Man, Scott P. France, Friedemann Leipold, Shahed Hussain, Laura Toca-
Gonzalez, Rebecca Marchington, Sam Hart, Johan P. Turkenburg, Gideon Grogan, and Nicholas J Turner
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b00782 • Publication Date (Web): 10 May 2016
Downloaded from http://pubs.acs.org on May 14, 2016
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Stereoselectivity and Structural Characterization of
an Imine Reductase (IRED) from Amycolatopsis
orientalis
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Godwin A. Aleku,a† Henry Man,b† Scott P. France,a Friedemann Leipold,a Shahed Hussain,a
a
a
Laura TocaꢀGonzalez , Rebecca Marchington , Sam Hart,b Johan P. Turkenburg,b Gideon
Groganb* and Nicholas J. Turnera*
aSchool of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131
Princess Street, Manchester, M1 7DN, UK.
bYork Structural Biology Laboratory, Department of Chemistry, University of York, YO10 5DD
York, UK.
ABSTRACT: The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4)
catalyzes the NADPHꢀdependent reduction of a wide range of prochiral imines and iminium
ions, predominantly with (S)ꢀselectivity and with e.e.s of up to >99%. AoIRED displays up to
100ꢀfold greater catalytic efficiency for 2ꢀmethylꢀ1ꢀpyrroline (2MPN) compared to other IREDs,
such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)ꢀselectivity, and thus
AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic
properties, with inversion of stereoselectivity observed between structurally similar substrates,
and also, in the case of 1ꢀmethylꢀ3,4ꢀdihydroisoquinoline, for the same substrate, dependent on
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the age of the enzyme after purification. The structure of AoIRED has been determined in an
‘open’ apoꢀform, revealing a canonical dimeric IRED fold in which the active site is formed
between the Nꢀ and Cꢀterminal domains of participating monomers. Coꢀcrystallisation with
NADPH gave a ‘closed’ form in complex with the cofactor, in which a relative closure of
domains, and associated loop movements, has resulted in a much smaller active site. A ternary
complex was also obtained by coꢀcrystallization with NADPH and 1ꢀmethylꢀ1,2,3,4ꢀ
tetrahydroisoquinoline [(MTQ], and reveals a binding site for the (R)ꢀamine product which
places the chiral carbon within 4 Å of the putative location of the C4 atom of NADPH that
delivers hydride to the C=N bond of the substrate. The ternary complex has permitted structureꢀ
informed mutation of the active site, resulting in mutants including Y179A, Y179F and N241A,
of altered activity and stereoselectivity.
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KEYWORDS: Biocatalysis; Chiral Amine; Imine Reductase; Oxidoreductase; NADPH
INTRODUCTION
The asymmetric synthesis of chiral amines is of central importance to the chemical industries,
as these moieties feature in a large proportion of pharmaceuticals and agrochemicals. Recently a
number of approaches have been reported for the biocatalytic synthesis of chiral amines,1
including the application of hydrolases,2 transaminases,3 amine oxidases4 and amine
dehydrogenases.5ꢀ7 The direct reduction of prochiral imines by NAD(P)Hꢀdependent
oxidoreductases, termed imine reductases or IREDs, is now receiving more attention, after
studies by Mitsukura and Nagasawa8ꢀ10 demonstrated that two strains of Streptomyces contained
NADPHꢀdependent oxidoreductases capable of stereocomplementary reductions of the model
substrate 2ꢀmethyl pyrroline (2ꢀMPN, 1a, Scheme 1).
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Since the initial identification of these (R)ꢀ and (S)ꢀselective IREDs, several groups have now
reported the isolation and application of further oxidoreductases from a wider subfamily that are
capable of the asymmetric reduction of prochiral imine substrates with a high degree of
stereoselectivity.11ꢀ17 The latest developments in this area has been summarized in three recent
reviews.18ꢀ20 The interest in IREDs has been stimulated by the possibility that the enzymes may
offer advantages of selectivity and environmentally benign methodology compared to abiotic
methods of asymmetric imine reduction that employ transition metal catalysts.21
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Scheme 1. Stereocomplementary reductions of 2ꢀMPN 1a by Streptomyces spp.
The mechanism of IREDs is not well understood and indeed it appears that different members
of the family employ different amino acid residues within the active site for substrate recognition
and/or catalysis. The first structure of an enzyme for which NADPHꢀdependent IRED activity
towards 1a had been demonstrated was the enzyme Q1EQE0 from Streptomyces kanamyceticus,
which catalyzes the (R)ꢀselective reduction of 1a albeit with low activity.16 The structure of this
IRED consists of an intimate homodimer in which the two active sites are located at the interface
between an Nꢀterminal Rossman domain of one subunit, and a Cꢀterminal helical bundle in the
other. The domains in each monomer are connected by a long helix from which an aspartate
residue, Asp187, protrudes into the active site, with its side chain approximately 8 Å from the C4
atom of the nicotinamide ring of NADPH that delivers and accepts hydride. The structure is
most similar to an hydroxyisobutyrate dehydrogenase 2CVZ,22 in which a lysine residue, which
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superimposed with the aspartate in Q1EQE0, was suggested to be the proton donor to the nascent
alcohol in the reduction of the titular substrate by that enzyme. By analogy, it was proposed that
Asp187 may act as a proton donor in the reduction of imine substrates, and indeed mutation of
this residue to Ala or Asn by ourselves16 and other groups13 in Q1EQE0 and homologs, resulted
in variants with reduced catalytic activity. Interestingly, in IREDs that possess (S)ꢀselectivity
towards 1a, the Asp/Lys position is occupied by a tyrosine residue, which in this case has its
phenolic hydroxyl group positioned approximately 5 Å from the NADPH C4 atom, possibly as a
proton donor, as has been demonstrated for the IRED pteridine reductase previously.23 The
location of this tyrosine was revealed using structures of (S)ꢀselective IREDs from
Streptomyces,12 Bacillus24 and Nocardiopsis24 and its mutation to Ala13 or Phe24 has again been
shown to diminish IRED activity in such enzymes.
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Recent work by Pleiss and Hauer13 has shown that there exists a much wider family of
potential IREDs, with a range of different amino acids in the Lys/Asp/Tyr position as predicted
by sequence alignments. Indeed Wetzl and coꢀworkers17 have recently shown that some of these
homologs contain residues with aprotic sideꢀchains, such as phenylalanine, in this position.
Despite the mutational investigations at this position, the absolute requirement for a proton donor
in IRED catalysis has not been clarified, as it may be that the imine substrate is already
protonated in solution prior to hydride delivery. We were prompted therefore to pursue structural
and mechanistic investigations of an IRED with an aprotic residue in the Lys/Asp/Tyr position,
and we selected the IRED homolog Uniprot code R4SNK4 (named AoIRED) from the
vancomycin producer Amycolatopsis orientalis, which features an Asn residue in this position.
Biotransformation and kinetics studies reveal AoIRED to be predominantly (S)ꢀselective, with
superior activity to previously described (S)ꢀIREDs, and also to possess surprising properties that
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include inversions of stereoselectivity upon very small changes in substrate structure, and also,
with four of the substrates, upon storage of the enzyme. In addition, structural investigations
have yielded both an apoꢀstructure and an NADPH complex, but also a ternary complex with a
reaction product, (R)ꢀ1ꢀmethylꢀ1,2,3,4ꢀtetrahydroisoquinoline [(R)ꢀMTQ]. The structures have
informed rational mutagenesis studies of AoIRED, but also suggest a significant role for protein
dynamics in catalysis by this enzyme.
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RESULTS
Substrate specificity and stereoselectivity
The gene encoding AoIRED was cloned and expressed in E. coli, and the protein purified using
protocols detailed in the Supporting Information (Section S2). Following purification, the effects
of pH and temperature on the activity of AoIRED using 1a as substrate were determined
(Supporting Information; Section S3). The pH optimum was found to be 7.5 and the temperature
optimum 50°C, although rapid denaturation was also observed at this temperature. In order to
characterize the activity of AoIRED with respect to substrate scope we examined a broad range
of cyclic imines. When stored at 4°C at a concentration of 2 mg mLꢀ1, the enzyme retained >90%
activity after 4 weeks. Examination of a series of 2ꢀsubstituted pyrrolines (1a-d), piperideines
(3a-g) and azepines (5a-b) (Scheme 2, Table 1) established that AoIRED catalyzes reduction of
the 2ꢀmethylꢀsubstituted derivatives (1a, 3a & 5a) predominantly to the corresponding (S)ꢀamine
products with excellent e.e.s of up to 98%.
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Scheme 2. Monocyclic prochiral imines reduced by fresh purified AoIRED.
Table 1. Monocyclic prochiral imines reduced by fresh purified AoIRED.
Substrate
Product
Conversion
(%)
e.e.
Absolute
(%)
Configuration
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n.d.[c]
95
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(S)ꢀ
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(ꢀ)[b]
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98
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>99
98
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>99
>99
>99
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>99
>99
>99
95
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>99
>99
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3g
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6b
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[a]Inversion of selectivity was observed when a 24 h old aliquot of AoIRED was used as the
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catalyst. Results of biotransformations using fresh purified AoIRED are presented. Absolute
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configuration not determined.[c] Lack of baseline separation prevented e.e. calculation; see
Figure S7.
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Interestingly, and in contrast to the selectivity pattern observed with the Streptomyces
IREDs10,15 the (S)ꢀamine products were also preferentially formed upon reduction of 2ꢀ
phenylpyrroline 1b (95% e.e.) and 2ꢀphenylpiperideine 3b (40% e.e.), which, due to a change in
CahnꢀIngoldꢀPrelog priority, represents an actual inversion of stereoselectivity, in which hydride
attacks the opposite face of the substrate. Remarkably, replacement of phenyl by a paraꢀ
fluorophenyl 3c or paraꢀmethoxyphenyl 3d substituent on the piperideine ring led to a reversal
of stereoselectivity, yielding (R)ꢀ4c (60% e.e.) and (R)ꢀ4d (90% e.e.) respectively. These results
suggest that very small changes in the substrate, in this case replacement of H by F or OMe, can
lead to dramatic changes in the stereoselectivity of reduction, presumably through associated
changes in the substrate binding mode. Interestingly, transformation of azepine 5a using fresh
purified AoIRED gave the (S)ꢀamine, as shown in Table 1, but using older enzyme, the
stereoselectivity was shown to invert, yielding the (R)ꢀproduct. This phenomenon is described in
more detail below.
Inversion of stereoselectivity in the reduction of 1-methyl-3,4-dihydroisoquinoline 9.
Further evidence of selectivity changes upon binding were observed when using
dihydroisoquinoline and βꢀcarboline substrates (Scheme 3, Table 2).
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Scheme 3. Bulky prochiral imines reduced by fresh purified AoIRED.
Table 2. Bulky prochiral imines reduced by fresh purified AoIRED.
Substrate
Product
Conversion
e.e. (%)
Absolute configuration
(%)
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100
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n/a
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>99
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[a] Inversion of selectivity was observed when a 24 h old aliquot of AoIRED was used as the
catalyst. Results of biotransformations using fresh purified AoIRED are presented. n/a= not
applicable.
Thus reduction of βꢀcarboline 13a yielded the (R)ꢀproduct (99% e.e.), albeit in low yield,
whereas reduction of 13c, which features a methoxy substituent on the phenyl ring, yielded the
(S)ꢀamine (79% e.e.).
Even more remarkable was the reduction of 1ꢀmethylꢀ3,4ꢀ
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dihydroisoquinoline 9, in which the stereoselectivity of reduction was found to depend upon the
form and age of the biocatalyst used. Thus a fresh lysate of cells expressing AoIRED converted 9
to (S)ꢀ10 (85% e.e.) as did fresh purified enzyme (81% e.e.). However, an aliquot of purified
AoIRED stored at 4°C for 24 h converted 9 to (R)ꢀ10 (98% e.e.) (Figure 1). Monitoring the
stereoselectivity as a function of storage time showed a steady increase in the preponderance of
(R)ꢀ10, eventually leading to total inversion of enantioselectivity towards 9 with an excellent e.e.
of 95ꢀ98% within 24 h and >99% after 14 d. Varying the reaction pH from 6ꢀ10 did not have any
effect on the selectivity change. Any contribution to the effect from the recycling enzyme
glucose dehydrogenase (GDH) was ruled out, as the inversion of stereochemistry was
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Figure 1. Selectivity switch of AoIRED towards 1ꢀmethylꢀ3,4ꢀdihydroisoquinoline 9. AoIRED
in whole cell, fresh lysate and fresh purified form yielded (S)ꢀ10, whereas older stocks of the
protein yielded (R)ꢀ10. (Experiments were repeated with three different batches of AoIRED and
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standard deviations are represented with error bars; On the yꢀaxis, ‘ꢀ100 % e.e. indicates 100%
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e.e. for the (S)ꢀenantiomer; ‘100 % e.e.’ indicates 100% e.e. for the (R)ꢀ.
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also observed when the reactions were performed with stoichiometric NADPH. The addition or
removal of imidazole, which was employed in purification, and may have interfered with
substrate binding, from the enzyme preparations, also did not have any effect, nor did the
addition of 1ꢀ20 mM DTT, added in an effort to slow the oxidation of active site thiols.
Attempts to reverse the selectivity of the older enzyme towards 9 were unsuccessful. An
inversion of stereoselectivity in the KRED from T. ethanolicus, reported by Phillips and coꢀ
workers,25 prompted us to study the stereoselectivity of reduction of 9 at different temperatures
by 7 day old enzyme, but no effect was observed. To investigate any peptideꢀassisted behavior of
AoIRED, we incubated the purified old stock of AoIRED with crude extract from E. coli bearing
an empty pETꢀ28a vector, using the same conditions for cultivation and induction as used in the
AoIRED wholeꢀcell system, but again, no effect was observed. However, the addition of equal
concentrations of bovine serum albumin (BSA) to the freshly purified AoIRED was shown to
slow the transformation from an (S)ꢀ to (R)ꢀselective enzyme. A 24 h old aliquot of purified
AoIRED containing BSA (5ꢀ10 times the concentration of AoIRED), added immediately after
purification, afforded (S)ꢀ10 with 76% e.e., whereas a 24 h old ‘AoIREDꢀonly’ aliquot yielded
(R)ꢀ10 (98% e.e.). Eventually, a 72 h old aliquot of ‘AoIRED + BSA’ challenged with 9 yielded
(R)ꢀ10 (e.e 98%).
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Stereoselective reduction of iminium ions
Tertiary chiral amines are important structural motifs in pharmaceuticals, and could in theory
be obtained through the IREDꢀmediated reduction of iminium ions. Indeed, recent reports by
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Zheng, Xu and coꢀworkers have demonstrated that (R)ꢀ and (S)ꢀselective IREDs from
Paenibacillus lactis catalyzed the reduction of indoline iminium ions with excellent
stereoselectivity.26, 27 The ability of AoIRED to catalyze the reduction of iminium ions was tested
using substrates 15a-15d, 17, 19a and 19b (Scheme 4, Table 3). Conversions ranged from 6%
for 15c to 90% for 19a, with good to excellent e.e.s, the best being achieved for substrates 15b
and 17. The highest conversion obtained was for Nꢀbenzylated substrate 15b and Nꢀ
methyldihydroisoquinoline 19a, with 79% and 90% respectively.
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Scheme 4. Prochiral iminium ions reduced by fresh AoIRED lysate.
Table 3. Prochiral iminium ions reduced by fresh AoIRED lysate.
Substrate
Product
Conversion
(%)
8
e.e (%)
Absolute
configuration
(R)ꢀ
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15b
15c
15d
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n.d.[b]
n.d.[c]
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(R)ꢀ
(R)ꢀ
(R)ꢀ
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19b
20a
20b
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(S)ꢀ [a]
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[c]
baseline separation prevented e.e. calculation; see Figure S19.
prevented e.e. calculation; see Figure S20.
Lack of baseline separation
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Kinetics
The determination of the kinetic parameters for representative substrates (Table 4) revealed an
increasing turnover number with increasing ring size from fiveꢀ (1a) to six membered (3a) rings
(1a, 3a). The kcat/Km values for 1a and 3a were 0.17 s−1 mMꢀ1 and 0.53 s−1 mMꢀ1 respectively,
which for 1a is approximately 100ꢀfold greater than the IRED from Streptomyces GF3546
(kcat/Km = 0.002 s−1 mMꢀ1),11 which also possesses (S)ꢀselectivity towards 1a, revealing AoIRED
to be the most catalytically efficient (S)ꢀselective IRED described in this class to date. The
highest turnover number, and indeed the highest catalytic efficiency, for AoIRED was observed
with the bicyclic dihydroisoquinoline 7 (kcat = 0.94 s−1; kcat/Km = 2.58 s−1 mMꢀ1) although the
superior binding affinity for a ligand was observed with the bulky 2ꢀphenylpiperideine 3b (Km =
0.22 mM).
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Table 4. Steadyꢀstate kinetic parameters for selected AoIRED substrates.
5
6
Substrate
Km (mM)
kcat (s−1)
kcat/Km (s−1ꢁmM−1)
7
8
9
0.87 ± 0.43
0.65 ± 0.11
0.22 ± 0.01
0.51 ± 0.16
0.36 ± 0.04
0.72 ± 0.05
0.96 ± 0.11
0.15 ± 0.08
0.35 ± 0.01
0.12 ± 0.00
0.14 ± 0.01
0.94 ± 0.01
0.74 ± 0.02
0.77 ± 0.02
0.17 ± 0.18
0.53 ± 0.11
0.38 ± 0.08
0.27 ± 0.07
2.58 ± 0.30
1.03 ± 0.40
0.80 ± 0.20
1a
3a
3b
3c
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10
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9
19b
Structural studies of AoIRED
The structure of AoIRED has been obtained in three forms: an ‘open’ apoꢀstructure, a ‘closed’
NADPH complex, and a ternary complex of the ‘closed’ form that also features (R)ꢀ10, the
product of (R)ꢀselective reduction of 2ꢀmethylꢀdihydroisoquinoline 9. AoIRED shares the
intimately associated domain sharing dimeric structure of other IRED structures in the database
(Figure 2).
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Figure 2. Dimer structure of AoIRED illustrating the canonical IRED fold with domain sharing
between subunits. Structure is shown in ribbon format with monomers A and B in light blue and
gold. NADPH is shown in cylinder format with carbon atoms in grey, is shown at the dimer
interface.
Indeed, analysis on the DALI server28 indicated that the closest structural homolog was the
(R)ꢀselective Q1EQE016 (3ZHB; 46% amino acid sequence identity; rmsd 2.3 Å over 282 Cꢀ
alpha atoms). The monomer structure adopts the canonical IRED fold now established for both
(R)ꢀ and (S)ꢀselective enzymes with a standard Rossman fold consisting of residues Thr2 to Gly
165; the transdomain helix (α8) from Leu166 to Ser194 and the Cꢀterminal helical bundle
formed from α8 and α9−α12, culminating in Arg290. A full representation of the secondary
structural motifs can be found in Figure S23. Superimposition of AoIRED with (R)ꢀselective
IRED Q1EQE0 and (S)ꢀselective BcSIRED revealed a very high level of conservation of
structure within the Rossman fold, and indeed within the Cꢀterminal helical bundle.
Superimposition of the apoꢀAoIRED with the AoIREDꢀNADPH complex revealed that the loop
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Thr225 to Gly236 between helices α9 and α10 displayed a remarkable 180° flip to bring the loop
from beneath the Cꢀterminal domain to occupy a position towards at the distal region of the
active site cleft (Figure S24), giving a ‘closed’ form of the active site that is clearly reduced in
volume compared to the actives site cleft in the apo structure. The loop movement in the
NADPH complex appears to result from a 180° flip of the peptide bond between Ala 223 and
Pro224. There are also substantial changes in the Rossman fold coincident with NADPH
binding: the region between Leu69 and Ala78 between strand β4 and helix α4 folds over the
Rossman domain with the result that the side chain of Tyr72 flips from the NADPH phosphate
binding pocket on the enzyme periphery in the apoꢀstructure, to the interior of the domain, where
the phenolic hydroxyl now forms hydrogen bonds with the side chains of Asp118, Tyr116 and
Ser96 (Figure S25). This moves the Cα and hydroxyl group of Tyr72 distances of 6 Å and 17 Å
respectively. The effect is to remove the side chain of Tyr72 from the ADP binding pocket
where it would occlude the binding of the adenine ring. Coincident with this large movement is
the reorientation of the side chain of Arg36 in the apoꢀstructure from the enzyme periphery to the
ADP binding pocket, where it now interacts with the ADP ribose 2’ phosphate. We have termed
the structure of the apoꢀ and NADPHꢀcomplex structures to be the ‘open’ and ‘closed’ forms of
AoIRED respectively.
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11
12
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Numerous approaches were taken to obtaining complexes of AoIRED with imine substrate or
amine products, including a range of ligands in combination with NADPH and NADP+. Coꢀ
crystallization of AoIRED with NADPH and racꢀ10 yielded a ternary complex that superimposed
well with the NADPH complex, with an rmsd for the subunit ‘A’ of 0.29 Å over 276 Cꢀalpha
atoms. The omit map at the subunit interface revealed density consistent with the presence of the
(R)ꢀenantiomer of MTQ, (Figure 3), and also for NADPH, excepting the nicotinamide riboside.
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The binding of NADPH has been sufficient to close the complex around the amine ligand and
this is now found parallel to the active site cleft, ideally positioned for hydride delivery from
NADPH to the electrophilic carbon. The plane of the ligand is stacked against hydrophobic
residues (A)Leu175 and (A)Met178 on one side of the active site, and (A)Pro124 on the other,
with the nitrogen atom of (R)ꢀMTQ at 3.4 Å from the amide of Asn241 and 4.4 Å from the
phenol group of Tyr179. The side chain of Asn171, homologous with the Asp and Tyr residues
of (R)ꢀ and (S)ꢀselective enzymes studied previously, is relatively remote from the amineꢀ
NADPH interaction, above the aromatic ring of (R)ꢀMTQ as presented in Figure 3 at a distance
of approximately 5.0 Å. Superimposition with the NADPH complex revealed that the chiral
carbon of the ligand would be ideally placed at approximately 3.5 Å from the C4 atom of the
nicotinamide ring of NADPH that delivers hydride to the electrophilic carbon of the C=N bond.
The identification of a possible ligandꢀbinding site in the ternary (R)ꢀMTQ complex prompted us
to mutate local residues in order to gauge their influence on catalytic activity.
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Figure 3. Detail of amine binding site in ‘closed’ AoIRED (R)ꢀMTQ ternary complex obtained
through coꢀcrystallization of AoIRED with racemic MTQ and NADPH. The peptide backbone
and side chain carbon atoms of monomers A and B are colored light blue and gold respectively.
Electron density corresponds to the Fo – Fc (omit) map contoured at a level of 2.5σ. (R)ꢀMTQ
atoms have been added for clarity. The omit map also features density for the ADPꢀriboseꢀ2’ꢀ
phosphate, but not the nicotinamide riboside, which could not be modelled. Atoms for the latter
have been included for illustrative purposes using superimposition with the NADP(H) complex
structure as a basis.
Site-directed mutagenesis of AoIRED
In order to probe the role of various activeꢀsite residues within AoIRED a series of siteꢀ
directed mutagenesis experiments was performed. The structure of AoIRED in complex with
(R)ꢀ10 suggested that some residues, such as Tyr179 and Asn241 within or near the
hydrophophobic pocket opposite the NADPHꢀbinding site may be involved in ligand
recognition, especially through interactions with the nitrogen atom. These interactions were
explored through the creation and assay of mutants Y179A, Y179F and N241A. The
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contribution to activity of residue Asn171, the pendant residue at the top of the active site as
pictured in Figure 3, and which corresponds to Asp187 in SkIRED was also assessed through
assay of mutants N171A and N171D.
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Y179A and Y179F were shown to be active against a broad panel of substrates, showing high
enantioselectivity in some cases, although the activity of the Y179A mutant was markedly lower
towards substrates 1a, 1b, 3b and 3d (selected data are presented in Table 5; for full details see
Table S5). Stereoselectivity towards smaller substrates such as 1a was not affected, but, notably,
enantioselectivity toward 2ꢀphenylpiperideine 3b and bicyclic imine 11 was inverted for both
Y179A and Y179F variants. The N241A mutant displayed reduced conversions to the WT for
smaller substrates, but broader substrate tolerance (Table 5; Table S6). Of particular note were
the reactions with substrates 11 and 13c, all of which gave higher conversions than the WT
enzyme, and with inverted stereoselectivity. In general, the N171A and N171D variants gave
lower conversions than the WT enzyme, reflecting lower activity, and largely with no change in
stereopreference. (Table 5; Table S7).
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Table 5. Biotransformation of selected substrates by active site mutants of AoIRED.
Data for AoIRED variants [C (%); e.e. (%) and abs. config. amine product]
7
8
Imine
WT
Y179A
Y179F
N241A
N171A
N171D
9
10
11
12
13
14
15
16
17
18
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20
21
22
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96; 82 (S)ꢀ 23; 99; (S)ꢀ 35; 47 (S)ꢀ 43; 81 (S)ꢀ
31; >99
(S)ꢀ
2; >99 (S)ꢀ
1a
100; 95
(S)ꢀ
8; 99 (S)ꢀ 99; 92 (S)ꢀ 43; 81 (S)ꢀ 100; 4, (S)ꢀ
100; 95
(S)ꢀ
1b
99; 40 (S)ꢀ 32; 43 (R)ꢀ 93; 8 (R)ꢀ
99; 0
41; 68 (S)ꢀ 80; 80 (S)ꢀ
3b
3d
11
70; 90 (R)ꢀ 3; 59 (R)ꢀ 25; 84 (R)ꢀ 56; 72 (R)ꢀ 2; 99 (R)ꢀ 11; 80 (R)ꢀ
50; 79 (S)ꢀ
15; >99
5; 99 (R)ꢀ 99; 85 (R)ꢀ 2; 31 (S)ꢀ
2; 99 (S)ꢀ
(R)ꢀ
15; 62 (S)ꢀ 10; 99 (S)ꢀ 9; 99 (S)ꢀ 96; 60 (R)ꢀ 18; 71 (S)ꢀ 6; 99 (S)ꢀ
13c
Kinetic studies of variants of Y179, N241 and N171 using substrates 1a, 7 and 9 (selected data
are presented in Table 6; further data in Table S8ꢀS10) showed that the Y179A variant displayed
up to 1.6 fold improved catalytic efficiency towards dihydroisoquinolines 7 and 9, compared to
the WT, although the Y179F variant was less efficient (Table 6). N241A displayed comparable
kinetics overall to the WT, with slight increases in Km. Analysis of N171A and N171D variants
revealed that mutations at N171 result in higher Km values and lower turnovers for bicyclic
substrates (Table 6; Table S10), contributing to lower secondꢀorder rate constants overall,
particularly for the alanine mutant.
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Table 6. Kinetic parameters of active site mutants of AoIRED against selected substrates
5
6
7
8
Kinetic data for AoIRED variants [Km (mM); kcat (s−1); kcat/Km
(s−1ꢁmM−1)]a
9
Imine
WT
Y179A
Y179F
N241A
N171A
N171D
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
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34
35
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0.87; 0.15;
0.17
1.77; 0.03;
0.05
0.87; 0.01;
0.02
1.27; 0.22;
0.17
7.90; 0.12;
0.02
2.22; 0.17;
0.01
1a
0.36; 0.94;
2.58
0.39; 1.52;
3.95
0.85; 0.32;
0.38
0.47; 0.95;
2.02
1.90; 0.15;
0.08
1.44; 0.15;
0.10
7
9
0.72; 0.74;
1.03
0.45; 0.92;
2.03
0.19; 0.07;
0.36
0.83; 0.76;
0.92
3.15; 0.01;
0.01
0.72; 0.21;
0.30
[a] Errors have been omitted from this Table for reasons of space, but can be found in relevant
Tables in the SI.
DISCUSSION
AoIRED is an active imine reductase with a broad substrate spectrum and the potential to serve
as a valuable asymmetric catalyst with certain substrates, including 5ꢀ (1b-1d), 6ꢀ (3a, 3d) and 7ꢀ
(5a) membered moncyclic imines, as well as isoquinolines such as 9 and some iminium ion
substrates. However, the situation is complicated by the observation with AoIRED, but also
other IREDs,17,24 of small changes in substrate structure having major changes on stereochemical
preference. AoIRED appears to display a mixed stereopreference for substrates that is difficult to
visualize, as CIP priority changes render the use of ‘reꢀ/siꢀ‘ and ‘R/S’ terminology confusing.
Scheme 5 shows that one set of substrates, exemplified by 1a, 2a and 3d are attacked by hydride
at the prochiral face of the molecule with the imine bond on the rightꢀhand side of the nitrogen
atom; conversely, the second set of substrates, represented by 3b, 3c and 9 (by stored AoIRED)
are attacked on the prochiral face with the imine bond on the leftꢀhand side of the nitrogen atom.
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Scheme 5. Two sets of prochiral imines representative of alternate stereochemical outcomes in
reductions by AoIRED. The NADPH hydride attacks compounds grouped A or B from the front
of the page in each case.
Even the very small changes in structure between 3d and 3b require that AoIRED must invert
the substrate to present the opposite face to the nicotinamide ring for the attack of hydride at the
electrophilic carbon of the C=N bond. This may implicate the use of more than one binding
mode, or even binding site, for different substrates. A multiplicity of binding modes is also one
possible explanation suggested by the remarkable inversion of stereoselectivity observed for the
reduction of substrate 9 on storage of the enzyme, and the plasticity of the active site suggested
by structural studies may also play a role in the complex behavior observed.
An extreme example of the unusual stereoselective properties of AoIRED is the inversion of
stereopreference in the reduction of 1ꢀmethylꢀ3,4ꢀdihydroisoquinoline 9. Both fresh lysate and
fresh purified AoIRED catalyzed the formation of the (S)ꢀamine product 10, but storage yielded
an enzyme that catalyzed the formation of the (R)ꢀ enantiomer. Despite many investigations, it
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did not prove possible to reverse this effect, although BSA was shown to slow the transformation
from an (S)ꢀ to an (R)ꢀ selective enzyme. BSA is known to stabilize the conformation of proteins
including dehydrogenases through hydrophobic effects29 and also to alter kinetic and
thermodynamic parameters through molecular crowding,30 although we are unaware of any
associated effects on the stereoselectivity of enzymes. Possible explanations for the observed
inversions of stereoselectivity could therefore include conformational changes or changes in
protein oligomerization on storage, but also possibly changes in the hydration of the active site
cleft, which have been observed by Phillips to invert the enantioselectivity of the KRED from T.
ethanolicus.31 Given the time scale of the conformational change, Xꢀray crystallographic studies
have not been able to shed light on the phenomenon of inverted stereoselectivity, but do provide
information on some of the mobile elements in AoIRED.
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The structures of AoIRED suggest that multiple conformations of the enzyme are indeed to be
encountered along the reaction coordinate, with major loop reorganisations observed on cofactor
binding. Some of this mobility, such as the movement of the tyrosine loop in the Nꢀterminal
domain, may be general, as this tyrosine is observed in all currently known IRED structures.
The loop movements beneath the active site in AoIRED, however, may be restricted to closely
related homologs, as there is clearly some diversity in IRED sequences in this region. This loop
movement, in conjunction with the relative closure of the two domains that participate in active
site formation, serves to close the active site to form a pocket that is much reduced in volume
compared to the active site cleft that has been observed in IRED structures previously, and in
which cofactor and substrate can interact for the reduction reaction. One abiding mystery of
IRED catalysis is the ability of stereocomplementary enzymes to catalyze the production of
antipodal amine products using apparently large active site clefts that appear to be fairly similar
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in nature. The observation of the smaller closed active site in the AoIRED ternary complex
suggests a ligand recognition site, at least in this enzyme, that is now small enough to possibly
exert control over which prochiral face of the imine is presented to the hydride.
10
11
12
13
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In the time taken to acquire pure AoIRED and to crystallize it, it is probable that the form of
the enzyme in crystallization drops is the (R)ꢀselective form, and, indeed it is the (R)ꢀ enantiomer
of 10 that is found in the ternary complex structure obtained by coꢀcrystallizing AoIRED with
NADPH and racemic 10. This complex placed cofactor, amine and active site in context, and
allowed a structureꢀguided approach to the possible roles of residues through mutational
analysis. Although N241 especially appears to interact directly with the nitrogen atom of the
amine product, it cannot act as a general acid in protonation of the reaction intermediate. Indeed
the imine substrate is probably protonated at the pH values used for bioconversions, so such
protonation may not be necessary. However, it may act as an anchor for the nitrogen atom that
fixes the ligand for reduction at the siꢀ face of the substrate in this case. Mutation of N241 to
alanine resulted in slight increases in the Km for 1, 7 and 9, and also created a more efficient
enzyme for the transformation of large substrates such as 11 and 13c, although its effect on
enantioselectivity was mixed, dependent on the substrate. In the same region, mutation of Y179,
which makes more distant contact with the amine nitrogen, to alanine actually created a more
efficient biocatalyst for the transformation of bicyclic imines. Together these residues may be
interesting targets for future mutational studies designed to expand the substrate spectrum of
AoIRED.
In the ternary complex structure Asn171, equivalent to Asp and Tyr residues previously
mooted to have roles in catalysis on the basis of homology to aldehyde dehydrogenase enzymes,
is somewhat remote from the interaction between cofactor and amine in the active site. While we
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cannot exclude a role in mechanism for the equivalent residue in other IREDs, certain homologs
such as AoIRED clearly exist which have no requirement for a protic residue in this position for
catalysis. Mutation of Asn171 to Asp certainly did not increase AoIRED activity and, indeed,
mutation to either Asp or Ala gave variants of higher Km and slightly altered stereoselectivity,
possibly suggesting a partial role for Asn171 in early substrate recognition in AoIRED as it
approaches the site of reduction.
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CONCLUSION
AoIRED represents an interesting new addition to the library of IRED catalysts for applications
in asymmetric synthesis. Previous studies suggest that these enzymes are applicable in either
wholeꢀcell reactions, in which substrate concentrations of up to 25 mM have been used,15 or as
isolated enzymes, for which established methods of nicotinamide cofactor recycling can be
applied.11 However, the studies of AoIRED again reveal that stereochemical control in these
enzymes is complex and the prediction of selectivity is especially difficult, and has to be
investigated on a caseꢀbyꢀcase basis. However, the description of a ternary complex of an IRED
does provide new avenues for mutation directed at altering the substrate scope of this enzyme,
and the mutations of Tyr179 and Asn241 suggests that this may be possible, with the increased
activity towards certain substrates engendered by this mutation. It is certain that further
structural and mechanistic studies will be required to shed more light on mechanism and
stereoselectivity and IREDs, and that the determinants of each may be different for subgroups of
IREDs bearing different amino acids within their active sites.
EXPERIMENTAL SECTION
Chemicals
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Commercially available chemicals and reagents were purchased from SigmaꢀAldrich (Poole,
Dorset, UK), Prozomix (Haltwhistle, Northumberland, UK), Alfa Aesar (Karlsruhe, Germany)
and Acros Organics (Geel, Belgium) unless stated otherwise. HPLC solvents were obtained from
SigmaꢀAldrich (Poole, Dorset, UK) or ROMIL (Waterbeach, Cambridge, UK) and GC gases
from BOC gases (Guildford, UK). Further general information is given in the Supporting
Information (Section S1). 2ꢀMethylꢀ1ꢀpyrroline (1a), 2ꢀmethylpiperidine (4a), harmaline (13c)
and 3,4ꢀdihydroisoquinoline (7) were purchased from SigmaꢀAldrich (Poole, Dorset, UK). 2ꢀ
Methylpyrrolidine (2a), 1ꢀmethylꢀ3,4ꢀdihydroisoquinoline hydrochloride (9), 1ꢀmethylꢀ6,7ꢀ
dimethoxyꢀ3,4ꢀdihydroisoquinoline (11) and 6,7ꢀdimethoxyꢀ1ꢀmethylꢀ1,2,3,4ꢀtetrahydroꢀ
isoquinoline hydrochloride (12) were sourced from Acros Organics (Geel, Belgium),
2ꢀphenylpyrrolidine (2b) was purchased from Apollo Scientific (Stockport, UK) and 1ꢀmethylꢀ
1,2,3,4 tetrahydroisoquinoline (10) was obtained from GlaxoSmithKline (Stevenage, UK).
Compounds 3aꢀ3d, 3g, 4aꢀ4d, 4g, 5a & 5b, 6a & 6b, 13a & 13b and 14a and 14b were
synthesised according to previously reported methods.11,11,15 Procedures for the preparation of
3e, 3f, 4e, 4f, 15a-15d, 16a-16d, 17, 18, 19a, 19b, 20a and 20b and their characterization data
are included in the Supporting Information (Section S4).
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Gene synthesis, cloning, expression and protein purification
General information on strains and plasmids, and details of gene design and cloning protocols
can be found in the Supporting Information (Section S2). The codonꢀoptimized gene sequence
encoding AoIRED was synthesized by Biomatik (Cambridge, Ontario, Canada) and subꢀcloned
into pET28aꢀ(+) vector using NdeI and XhoI restriction sites to form pET 28aꢀHisꢀAoIRED
plasmid (Figure S1). Siteꢀdirected mutants of AoIRED were generated using the Stratagene
QuikChange site directed mutagenesis protocol, using primers as listed in the Supporting
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Information (Section S2). The presence of mutations in all constructs was verified by
sequencing. Production of AoIRED was achieved in E. coli BL21 (DE3) which had been
transformed with the recombinant AoIRED plasmid. Cultivation was performed in 500 mL
lysogeny broth (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with kanamycin (30
ꢂg ꢁmLꢀ1) added as the antibiotic marker. Cultures were initially incubated at 37°C with shaking
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at 250 r.p.m. At an optical density (OD600nm) of between 0.6 and 0.8, isopropyl βꢀDꢀ1ꢀ
thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM to induce the
expression of AoIRED. Incubation was continued at 30°C and 250 r.p.m. for 18 h. Cells were
then harvested by centrifugation and resuspended in sodium phosphate buffer (100 mM, pH 7.5).
Celles were disrupted using a Soniprep150 ultrasonicator (MSE, London, UK), employing 3 x 30
s bursts with 30 s intervals at 4°C. Cell debris was removed by centrifugation, after which the
resulting supernatant was loaded onto a 5 mL HisꢀTrap HP column (GE healthcare). The protein
was eluted using a gradient of 0ꢀ300 mM imidazole in sodium phosphate buffer (100 mM, pH
7.5) containing 300 mM NaCl. The purified enzyme was desalted using 30 kDa cutꢀoff Centricon
filters (15 mL), and washed twice with sodium phosphate buffer (100 mM, pH 7.5) to remove
NaCl and imidazole. For protein for crystallisation, further purification by size exclusion
chromatography (SEC) using a HiLoad 16/60 Superdex 75 PrepGrade (GE Healthcare) gel
filtration column was performed. AoIRED was purified by SEC with TrisꢀHCl buffer (50 mM,
pH 8.0) containing 500 mM NaCl. SEC trace (Figure S2) and SDSꢀPAGE analysis (Figure S3)
can be found in the Supporting Information (Section S2). The protein concentration was
determined using Bradford assay against BSA as a concentration standard.
Biotransformations
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Whole cell biotransformation reactions typically contained E.coli BL21(DE3) resting cells
containing expressed AoIRED (OD600nm of 50), 2ꢁ% (v/v) dimethylformamide, 5 mM imine
substrate and 50 mM glucose in sodium phosphate buffer (100mM, pH 7.5). Reactions were
incubated at 30°C with shaking at 250 r.p.m. for 18 h.
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Biotransformations using isolated enzymes were performed with purified AoIRED employing
glucose dehydrogenase (GDH)/NADPH as a cofactor recycling system. A typical 500 µL
reaction mixture contained 20 mM D
ꢀglucose, 0.2 mg mLꢀ1 GDH, 0.3 mM NADP+, 0.4 mg mLꢀ1
AoIRED 5 mM imine/iminium ion substrate and 2% (v/v) dimethyl formamide. The reaction
volume was made up to 500 µL with sodium phosphate buffer (100mM, pH 7.5). Reactions were
incubated at 30°C with shaking at 250 r.p.m. shaking for 18 h, after which they were quenched
by the addition of 30 µL of 10 M NaOH and extracted twice with 500 µL tertꢀbutyl methyl ether.
The organic fractions were combined and dried over anhydrous MgSO4 and analyzed on HPLC
or GC using chiral columns. Details of columns and analytical methods, with chromatograms,
can be found in the Supporting Information (Section S5).
Enzyme Assays
The imineꢀreducing activity of AoIRED was measured by monitoring the oxidation of NADPH
using a Cary 300 UVꢀVis spectrophotometer (Agilent, Santa Clara, CA, USA). The
consumption of NADPH was monitored at 340 (ε=6.22 mM−1ꢁcm−1) or 370 nm (ε=2.216
mM−1ꢁcm−1). The assay mixture in sodium phosphate buffer (100mM, pH 7.5) contained an
imine substrate in concentrations of up to 30 mM, NADPH at concentration of 160 ꢂM (340 nM)
or 300 ꢂM (370 nm), 1ꢁ% (v/v) DMSO and 10ꢀ50 ꢂg of pure enzyme, in a total volume of
1000 ꢂL. Control reactions without substrate featuring the same reaction conditions were run to
determine the background consumption of NADPH. The reaction was initiated upon the addition
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of purified AoIRED to the mixture. A unit of AoIRED was equal to the amount of the pure
enzyme required to consume 1 ꢂmol NADPH/ NADP+ per min. Activity measurements were
performed in triplicate and kinetic constants were determined through nonlinear regression based
on Michaelis–Menten kinetics (QtiPlot software).
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Protein Crystallization
Pure AoIRED was subjected to crystallization trials using a range of commerciallyꢀavailable
screens in 96ꢀwell sittingꢀdrop format, in which each drop consisted of 150 nL protein and
150 nL of precipitant reservoir solution. The best hits for apoꢀAoIRED were found in conditions
containing 0.2 M calcium acetate, 0.1 M Tris pH 9.0, 8% (w/v) PEG 550 monomethyl ether
(MME) and 8% (w/v) PEG 20K with protein at a concentration of 20 mg mLꢀ1. Larger crystals
for diffraction analysis using optimized conditions were prepared using the hangingꢀdrop method
in 24ꢀwell plate Linbro dishes with 2 ꢀL drops consisting of 1:1 ratio of mother liquor to protein.
The best crystals were obtained in conditions containing 0.2 M calcium acetate, 0.1 M Tris pH
9.0, 8% (w/v) PEG 550 MME and 8% (w/v) PEG 20K with protein at a concentration of 20 mg
mLꢀ1.
Coꢀcrystallization of AoIRED with NADPH (10 mM) did not yield crystals under the same
buffer conditions. In order to obtain the NADPH complex of AoIRED, native crystals were
transferred from the growth drop into a cryogenic solution which consisted of the mother liquor
containing ethylene glycol [20% (v/v)] and NADPH (10 mM), and incubated for 5 min, after
which they were immediately flashꢀcooled in liquid nitrogen.
Crystals of AoIRED complexed with (R)ꢀ10 in the ‘closed’ conformation were obtained by
subjecting pure AoIRED to crystallization trials using a range of commercially available screens
in sittingꢀdrop format with 1 mM NADPH and 1 mM racꢀ10 added into the mother liquor 1 h
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prior to dispensing 150 nL protein and 150 nL of precipitant reservoir solution. The best hits for
AoIRED complexed with (R)ꢀ10 were found in conditions containing 0.2 M lithium sulfate, 0.1
M BisTris pH 6.5, 25% (w/v) PEG 3350, 1 mM NADPH and 1 mM racꢀ10 with protein at a
concentration of 25 mg mLꢀ1. Larger crystals for diffraction analysis using optimised conditions
were prepared using sittingꢀdrop format 48ꢀwell MRC MAXI crystallization plates with 1 ꢂL
drops consisting of 1:1 ratio of mother liquor to protein. The best crystals were obtained in
conditions containing 0.25 M lithium sulfate, 0.1 M BisTris pH 6.5, 23% (w/v) PEG 3350, 1 mM
NADPH and 1 mM racꢀ10. The AoIRED complex with (R)ꢀ10 in the ‘closed’ conformation was
obtained by soaking the preꢀcomplexed crystals with compound NADPH and racꢀ10 for 5 min
prior to flashꢀcooling with liquid nitrogen without the addition of further cryoprotectants.
All crystals were tested for diffraction inꢀhouse using a Rigaku Micromaxꢀ007HF fitted with
Osmic multilayer optics and a Marresearch MAR345 imaging plate detector. Those crystals that
diffracted to a resolution of equal to, or better than, 3 Å resolution were retained for dataset
collection at the Diamond Light Source synchrotron.
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Data Collection, Structure Solution, Model Building and Refinement
Complete datasets described in this report were collected at Diamond Light Source, Didcot,
Oxfordshire, U.K. The apoꢀAoIRED dataset was collected on beamline I03, the NADPH
complex on beamline I04 and the ‘closed’ (R)ꢀMTQ complex on beamline I02 utilizing 10%
beam transmission and helical data collection.32 Data were processed and integrated using XDS33
and scaled using SCALA34 included in the Xia2 processing system.35 Data collection statistics
are given in the Supporting Information in Table S4. The crystals of apoꢀAoIRED in the ‘open’
form were in space group C2221. The crystals of the ‘closed’ complexes were in group P212121.
The structure of AoIRED was solved with MOLREP36 using a monomer model of the IRED
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