Two-Enzyme Hydrogen-Borrowing Amination of Alcohols Enabled by a Cofactor-Switched Alcohol Dehydrogenase

Article abstract of DOI:10.1002/cctc.201701092

The NADPH-dependent secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeSADH), displaying broad substrate specificity and low enantioselectivity, was engineered to accept NADH as a cofactor. The engineered TeSADH showed a >10 000-fold switch from NADPH towards NADH compared to the wildtype enzyme. This TeSADH variant was applied to a biocatalytic hydrogen-borrowing system that employed catalytic amounts of NAD⁺, ammonia, and an amine dehydrogenase, which thereby enabled the conversion a range of alcohols into chiral amines.

Full text of DOI:10.1002/cctc.201701092

Accepted Article
Title: Two-enzyme hydrogen-borrowing amination of alcohols enabled
by a cofactor switched alcohol dehydrogenase
Authors: Nicholas John Turner and Matthew Thompson
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10.1002/cctc.201701092
ChemCatChem
COMMUNICATION
Two-enzyme hydrogen-borrowing amination of alcohols enabled
by a cofactor switched alcohol dehydrogenase
Matthew P. Thompson [a] and Nicholas J. Turner *[a]
Abstract:
The
NADPH-dependent
secondary
alcohol
described AmDH from Exiguobacterium sibiricum.[10] Their
approach addressed the limitation of using
enantiocomplementary ADHs, but at the considerable expense
of substrate scope, which was limited to simple aliphatic
alcohols. Accordingly, there remains a need to further improve
this biocatalytic hydrogen-borrowing method for the asymmetric
amination of a broad range of alcohols with ammonia.
dehydrogenase from Thermoanaerobacter ethanolicus (TeSADH),
displaying broad substrate specificity and low enantioselectivity, has
been engineered to accept NADH as a cofactor. The engineered
TeSADH shows a >10,000-fold switch from NADPH towards NADH
compared to the wild type enzyme. This TeSADH variant has been
applied to a biocatalytic hydrogen borrowing system that employs
catalytic amounts of NAD+, ammonia and an amine dehydrogenase
(AmDH) thereby enabling the conversion a range of alcohols into
chiral amines.
Our aim was to develop a second-generation hydrogen-
borrowing system that combined the broad substrate scope of
our first generation process with the elegance of a single, non-
selective enzyme for alcohol oxidation. In order to realize this
goal, a number of challenges needed to be simultaneously
addressed (Figure 1b). The ADH employed must possess both
low enantioselectivity and broad substrate scope for the
oxidation of a wide range of alcohols. Furthermore the ADH
needed to accept NADH as cofactor to be compatible with the
cofactor dependence of engineered AmDHs. Finally,
thermostability, low Michaelis constants towards substrate and
cofactor, and ease of purification/handling would be highly
advantageous for application in hydrogen-borrowing cascades.
The activation of primary and secondary alcohols under mild
conditions, to allow subsequent nucleophilic substitution, has
been identified as a key area for method development in
synthetic organic chemistry.[1] In this context the development of
strategies based on hydrogen-borrowing is attractive because in
principle the only by-product is water.[2] Consequently there has
been considerable activity in this field and a number of elegant
redox self-sufficient (hydrogen-borrowing) reactions, using
ruthenium and iridium catalysts, have been developed.[3] Despite
the impressive advances in terms of substrate scope and
application, the requirement for high temperatures and high
catalyst loading, as well as a general lack of chemo- and
stereoselectivity, currently limit the application of these methods.
Biocatalytic amination of alcohols through enzyme cascades
employing ω-transaminases have been reported, although these
systems typically require several equivalents of a sacrificial
amine donor in order to driving the reactions to high
conversion.[4–6] A recent report by Lavandera and co-workers
described the use of stoichiometric diamines as the amine donor
in an efficient laccase/TEMPO coupled reaction for amination of
alcohols.[7]
Figure 1. a) General scheme for the hydrogen-borrowing amination of an
alcohol 1 to the corresponding primary amine 3 by employing an alcohol
dehydrogenase and amine dehydrogenase cascade reaction. b) The first
hydrogen-borrowing system relied on the use of enantiocomplementary ADHs
to achieve the alcohol to ketone conversion. However, it is desirable to
accomplish this with a single non-selective ADH. ADHs with broad substrate
scope and low enantioselectivity are known but are exclusively NADPH-
dependent. Protein engineering allows access to NADH-dependence by
rational design.
Recently we reported a biocatalytic hydrogen-borrowing
cascade in which a pair of enantio-complementary alcohol
dehydrogenases (ADHs) were coupled with an amine
dehydrogenase (AmDH) in order to achieve the conversion of
racemic secondary alcohols to enantiomerically pure chiral
amines on a preparative scale (Figure 1a).[8,9] This initial report
established a key proof-of-concept but also highlighted some
limitations, notably the requirement for
a
pair of
enantiocomplementary ADHs to achieve the alcohol oxidation
step. Following our first generation system, Xu and co-workers
described a dual enzyme approach using a single non-selective
ADH from Streptomyces coelicolor (ScCR) and
a newly
[a]
M. P. Thompson, Prof. N. J. Turner
School of Chemistry, Manchester Institute of Biotechnology
The University of Manchester
131 Princess Street, Manchester, M1 7DN (UK)
nicholas.turner@manchester.ac.uk
Supporting information for this article is given via a link at the end of
the document.
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Although many ADHs have been previously characterised,
only a limited number of wild-type enzymes have been reported
to show the broad substrate scope and low selectivity that was
required.[11,12] A single NADH dependent racemase has been
described but the substrate scope and reaction parameters have
not been explored.[13,14] Interestingly, Musa and co-workers
recently reported engineered variants of the NAD(P)H
S2–S5). From the data it was apparent that in the cases of R200
and G198, substitution for aspartate occurred with similar or
greater frequency than the analogous amino acid in TeSADH. At
position S199, the distribution of residues is broader, however
mutation of hydrophobic residues such as leucine appeared in
approximately 35 % of the sequences.
dependent
ADH
from
Thermoanerobacter
ethanolicus
(TeSADH) that showed low enantioselectivity in the reduction of
phenylacetone.[15–17] TeSADH meets many of the required
criteria with respect to selectivity, substrate scope, stability and
ease of handling. However, TeSADH exhibits very high
selectivity towards NADPH and hence it was envisaged that
significant protein engineering would be required to improve the
activity with NADH. Switching the cofactor specificity of
oxidoreductases has been accomplished using a variety of
approaches including using structural information to elucidate
beneficial mutations, and the use of multiple sequence
alignments of enzyme homologues that display differing cofactor
specificity.[18–20] The strategies described above have been used
to engineer the cofactor specificity in a number of enzymes;
often with an overall loss in catalytic efficiency.[21–24] Recently,
Arnold and co-workers reported an impressive general approach
(CSR-SALAD) for engineering cofactor specificity and
subsequent recovery of activity by screening targeted semi-
Figure 2. Ribose phosphate of NADP in TeSADH, labelled residues are
proposed to play a role in cofactor discrimination.
rational libraries.[25] As
a
complementary approach, we
The most commonly occurring single variants were
generated using the non-stereoselective W110A variant as a
template, and the relative activity with NAD vs. NADP was
compared. (Table 1) Both the variants R200D and Y218F were
inactive towards 4-methyl-2-pentanol.
considered taking advantage of the natural diversity in the
cofactor-binding domain amongst structural homologues of
TeSADH, to provide a basis for rational re-engineering of
cofactor specificity. Herein we describe
a structural and
sequence guided approach to affect a switch in the cofactor
specificity and subsequent application to
hydrogen-borrowing cascade.
a two-enzyme
Table 1. Specific activities of TeSADH variants for alcohol oxidations using
both NAD and NADP.
Since crystal structures of the non-selective variants of
TeSADH have not been reported we used the structure of the
wild-type enzyme as the basis for engineering cofactor
specificity. Examination of the crystal structure of TeSADH
(PDB: 2NVB) revealed a number of residues that are potential
points of contact for the ribose phosphate of NADP.[26] Figure 2
highlights residues (G198, S199, R200, and Y218) that are
proposed to be important for NAD/NADP discrimination in
TeSADH.
Entry
Variant
Specific
activity
Specific activity
Specificity
for NAD
vs. NADP
NAD[b]
NADP[a]
1
2
3
4
5
6
7
W110A (parent)
W110A/R200D
W110A/R200V
W110A/Y218F
W110A/S199L
W110A/G198D
237.06 ± 7.96
n.d.
1.79 ± 0.07
n.d.
1
14.30 ± 2.77
n.d
2.11 ± 0.11
n.d.
19
In the 3DM™ public database of ADHs, TeSADH belongs to
the 3FPLA subfamily containing 455 protein sequences (see
Supporting information). This allowed for expedient comparison
of TeSADH sequence to the broader subfamily. Wild-type
TeSADH possesses a tryptophan residue at position 110 and
Phillips and co-workers have shown that mutations at this
position confer low enantiospecificity towards a range of
secondary alcohols.[15–17,27–29] Inspection of the data revealed
approximately half of the sequences featured different residues
at position 110. The alignments were therefore constrained to
the 226 sequences that contained a conserved Trp-110. Using
the automatically generated 3DM sequence alignments we
examined the amino acid distribution at each of the four putative
positions (G198, S199, R200 and Y218) proposed to be involved
in NAD/NADP discrimination (Supporting Information, Figures
48.66 ± 2.77
1.01 ± 0.09
23.71 ± 1.18
2.73 ± 0.03
100.31 ± 3.31
50.50 ± 0.02
7
13153
282
W110A/G198D/S
199L
[a] Specific activity in mU mg-1 protein measured at 25 °C with 20 mM methyl
isobutyl ketone, 1 mM NAD. [b] As for NAD except using 1 mM NADP+ [b] Not
determined – specific activity < 0.5 mU/mg.
Remarkably however, the single point mutation G198D
simultaneously gave a more than 50-fold improvement in activity
towards NAD together with a ca. 200-fold reduction in activity
towards NADP, equating to >104-fold switch in specificity
towards NAD compared to the parent (Table 1, Entry 6).
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Despite a single mutation in TeSADH affording high activity
with NAD, we considered whether it would be possible to further
improve the activity this variant. It has been shown that
mutations around the adenine moiety of the cofactor can play an
important role in improving activity and even altering substrate
specificity.[30,31] Given the previous observation that the mutation
Y218F rendered TeSADH inactive, this site was excluded and
residues G244, N245, I248 were selected as candidates for
single-site saturation mutagenesis (Figure 3). The libraries were
generated using TeSADH W110A/G198D as a template, and
Next, we examined the performance of these engineered
variants of TeSADH in our hydrogen-borrowing cascade (Figure
1). Using the ChiAmDH[28] under conditions similar to those
previously reported,[4] we performed the hydrogen-borrowing
amination of 4-phenyl-2-butanol at [S] = 20 mM resulting initially
in conversions that were modest but encouraging (ca. 30 %). In
order to rapidly optimize conversions we took advantage of
surface response methods to probe a large space of reaction
[32]
conditions with a relatively low number of experiments.
(Supporting Information Table S2–S3 and Figure S7). The
reactions were optimized for reaction temperature, cofactor
concentration and relative loadings of TeSADH and AmDH.
Under these conditions conversions up to 86 % were obtained
employing the W110A/G198D variant. Despite their lower
catalytic efficiency similar results could also be obtained with the
remaining variants (Supporting Information Table S4).
screened
by
monitoring
the
formation
of
NADH
spectrophotometrically for the oxidation of racemic 4-methyl-2-
pentanol (Supporting Information Figure S6) resulting in the
identification of a number of clones with increased activity.
Using the optimized reaction conditions the cascade was
performed using TeSADH W110A/G198D against a panel of
alcohols 1a–12a (20 mM) representative of the broad substrate
scope of our first generation system (Table 3). The substrates
were aminated with excellent conversions of up to 90 %.
Table 3. Substrates, conversions and ee for the alcohols used in the
hydrogen-borrowing amination with newly engineered, NADH-dependent
TeSADH W110A/G198D and an AmDH.
Figure 3. Residues proposed to play a role in stabilising the Adenine moiety
of NADP in TeSADH.
The parent (TeSADH W110A/G198D) was the best
performing variants in the G244NNK pool. The N245NNK and
I248NNK pools revealed mutations to valine in the most active
clones. (Supporting Information Table S1). The newly identified
triple variants (TeSADH W110A/G198D/N245V and TeSADH
W110A/G198D/I248V) were overexpressed and purified. Their
kinetic parameters were determined and compared to both
previous TeSADH variants. (Table 2)
Entry
Substrate
Product distribution (%)[b]
ee (%)[b]
[a]
Alcohol
Ketone
Amine
1
2
3
4
5
6
7
8
rac-1a
rac-2a
rac-3a
rac-4a
rac-5a
rac-6a
rac-7a
rac-8a
rac-9a
10a
8
7
1
6
10
1
13
7
4
5
7
85
66
75
72
70
71
90
47
48
89
81
11
87 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
99 (R)
94 (R)
n.a.
The triple mutant TeSADH W110A/G198D/N245V displayed
an improvement in Km with NAD of more than 20-fold over the
parent TeSADH W110A, however, this was accompanied by a
decrease in the kcat, leading to a lower second-order rate
constant overall. (Table 2). Therefore the best variant was the
W110A/G198D that showed a more than 90-fold improvement in
second-order rate constant compared to the parent.
33
19
18
29
16
3
49
47
4
Table 2. Kinetic parameters of TeSADH variants for alcohol oxidation using
NAD
9
10
11
12
Entry
Variant [a]
kcat (s-1)
KM (mM)
11a
rac-12a
4
82
15
7
n.a.
>99 (R)
1
2
3
4
W110A (parent) [b]
W110A/G198D
1.60 ± 0.15
7.60 ± 0.37
1.93 ± 0.35
3.65 ± 0.46
19.22 ± 3.09
1.03 ± 0.11
0.44 ± 0.21
1.41 ± 0.34
[a] Analytical scale reactions (500 µL) performed in NH4Cl (1M, pH 9) with
substrates (20 mM), NAD+ (3 mM), ChiAmDH (2 mg/mL) and TeSADH
W110A/G198D (0.6 mg/mL). [b] Conversion and enantiomeric excess
determined by GC analysis – see Supporting Information
W110A/G198D/N245V
W100A/G198D/I248V
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Finally, to demonstrate the applicability of our second-generation
system, the hydrogen-borrowing cascade with TeSADH
W110A/G198D was performed on semi-preparative scale (100
mg). Substrates rac-1a and rac-4a were chosen owing to the
potential use of the amine products as synthetic intermediates
for (R,R)-dilevalol and (R,R)-formoterol respectively. The
asymmetric amination of rac-1a and rac-4a was achieved with
isolated yields of 69 and 84% respectively.
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the Royal Society for a Wolfson Research Merit Award.
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Keywords: biocatalysis
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Designed to share: Rational
engineering of the cofactor
dependence in a non-selective
alcohol dehydrogenase opens
the door to a second
Matthew P. Thompson,
Nicholas J. Turner*
Page No. – Page No.
Title
generation of hydrogen-
borrowing enzyme cascades
for the amination of alcohols
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