The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides

Article abstract of DOI:10.1002/anie.201804592

Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP-dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical-type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides.

Full text of DOI:10.1002/anie.201804592

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Accepted Article
Title: Aryl acid specificity of the amide bond synthetase McbA suggests
broad potential for the biocatalytic synthesis of amides
Authors: Gideon Grogan, Mark Petchey, Anibal Cuetos, Benjamin
Rowlinson, Stephanie Dannevald, Amina Frese, Peter Sutton,
Sarah Lovelock, Richard Lloyd, and Ian Fairlamb
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10.1002/anie.201804592
Angewandte Chemie International Edition
COMMUNICATION
Aryl acid specificity of the amide bond synthetase McbA
suggests broad potential for the biocatalytic synthesis of amides
Mark Petchey,[a] Anibal Cuetos,[a] Benjamin Rowlinson,[a] Stephanie Dannevald,[a] Amina Frese,[a] Peter W. Sutton,[b]† Sarah
Lovelock,[b]‡ Richard C. Lloyd,[b] Ian J.S. Fairlamb[a]* and Gideon Grogan[a]*
ATP-dependent amide bond synthetases (ABSs) have
been discovered in secondary metabolite pathways leading to
compounds such as novobiocin,10 cloromycin11 and
coumermycin.12 The enzymes NovL and CouL, for example,
couple the aminoflavone 1 with carboxylic acids 2 and 4 in their
respective biosynthetic pathways to give amide products 3 and 5
(Scheme 1A).
Abstract: Amide bond formation is one of the most important
reactions in pharmaceutical synthetic chemistry. The
development of sustainable methods of amide bond formation,
including those that are catalyzed by enzymes, is therefore of
significant interest. The ATP-dependent amide bond synthetase
(ABS) enzyme McbA, from Marinactinospora thermotolerans,
catalyzes the formation of amides as part of the biosynthetic
pathway towards the marinacarboline secondary metabolites.
The reaction proceeds via an adenylate intermediate, with both
adenylation and amidation steps catalyzed within one active site.
In this study, McbA has been applied to the synthesis of
pharmaceutical-type amides from a range of aryl carboxylic
acids with partner amines provided at 1-5 molar equivalents.
The structure of McbA has revealed the structural determinants
of aryl acid substrate tolerance and differences in conformation
associated with the two half-reactions catalyzed. The catalytic
performance of McbA, coupled with the structure, suggest that
this and other ABS enzymes may be engineered for applications
in the sustainable synthesis of pharmaceutically relevant (chiral)
amides.
The formation of the amide bond is one of the most important
reactions in pharmaceutical synthetic chemistry, accounting for
up to 16% of all synthetic steps in medicinal chemistry
laboratories.1 Amide bond synthesis typically involves the
activation of the carboxylic acid, using either toxic chlorinating
agents, or atom-inefficient coupling reagents,2 and so there is an
urgent need to investigate methods of amide bond formation that
are sustainable in terms of reagent safety and atom efficiency.
Enzymatic methods of amide bond synthesis that display these
advantages are appealing therefore.3,4 The amide bond is
encountered in secondary metabolites that are synthesised by
non-ribosomal peptide synthases (NRPSs).5 In this case, the
formation of the amide is complex, requiring ATP-dependent
formation of an intermediate adenylate of the carboxylic acid
substrate, followed by transfer to a phosphopantetheine thiol
enabled by an acyl/peptidyl carrier protein (ACP or PCP),
followed by condensation with the amine substrate, each
process taking place in a separate domain of a large, multi-
domain NRPS enzyme. While NRPS-mediated amide bond
formation may have a role in synthetic biology pathways towards
amides,6 their structural and catalytic complexity militates
against their application in preparative in vitro biocatalysis.
Hydrolases, such as N-acylases7 and lipases,8 have been
employed for amide bond formation, and are attractive in terms
of their simplicity and efficiency, but often require ester
Scheme 1. A: Amide bond synthesis by ATP-dependent amide bond
synthetases (ABSs) NovL and CouL involved in secondary metabolism in
actinomycetes; B: Formation of marinacarbolines such as 6a from -carboline
acid 6 and amine a catalyzed by McbA from Marinactinospora thermotolerans.
These enzymes are apparently capable of catalyzing
formation of the adenylate, but also of binding the amine for the
coupling reaction within the same active site, with no
involvement of separate acyl carrier protein or condensation
domains. A further enzyme, McbA, has recently been reported
in the biosynthetic pathway towards the marinacarbolines in
Marinactinospora thermotolerans by Ji and co-workers.13,14
McbA catalyzes the ATP-dependent coupling of the -carboline
derivative 6 with 2-phenylethylamine a to form 6a via adenylate
7 (Scheme 1B), but, unlike other ABSs, McbA also accepted
other amines, including substituted 2-phenylethylamines and
tryptamines, indicating some promise for the application of this
enzyme to reactions with a wider substrate spectrum.14 These
studies prompted us to further examine the activity of McbA to
include a range of -carboline, and other aryl carboxylic acid
acceptors. We have also determined the structure of McbA in
complex with AMP and 6, which has revealed the substrate
binding interactions within the active site, and may serve as a
platform for protein engineering to expand the substrate
specificity of this and related ABS enzymes.
substrates, and suffer from poor substrate scope.
More
recently, Flitsch and co-workers have recruited the adenylate-
forming ability of a carboxylate reductase (CAR) adenylation-
domain-plus-peptidyl carrier protein (CAR-A-PCP) to the
formation of amides, although a 100-fold excess of amine was
required to drive reactions to high conversions.9
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10.1002/anie.201804592
Angewandte Chemie International Edition
COMMUNICATION
Table 1. ATP-dependent amide couplings by McbA. Values represent %
conversions to amide products after 24 h as determined by HPLC analysis
McbA was expressed from a synthetic gene and the
protein purified using IMAC followed by gel filtration (Figure S1,
S2). A sample of the pure enzyme (1 mg mL-1; 9.4 nmol) was
applied to the coupling of 0.4 mM acid 6 and 0.6 mM 2-
phenylethylamine a, in the presence of 2 mM ATP on an
analytical scale, and full conversion to product amide 6a was
observed within 1 h using HPLC. Standard amide product was
prepared by reaction of the -carboline ester with the amine in
DMSO at 90°C (Supporting Information). We then further
explored the constraints on substrate structure in the McbA-
catalyzed reaction, but, in contrast to previous work,14 we
focused on the structure of the carboxylic acid acceptor
(Scheme 2).
Amine
Acid
a
b
c
d
6
8
100
96
82
100
6
81
100
0
6
0
0
0
0
26
17
0
9
10
11
91
0
0
0
McbA catalysed the formation of 6a and 6b with 100% and 81%
conversion respectively, confirming the findings of Ji and co-
workers.14 However, McbA also catalysed the coupling of 6 to
the racemic chiral amines c and d to form 6c and 6d, although
with lower conversions of 6% and 26%. Interestingly, analysis of
the product 6d by chiral HPLC revealed it to be predominantly of
the (S)-configuration, and with an e.e. of 96% (Figure S3),
indicating that McbA is capable of performing kinetic resolution
reactions. Tryptamine b was also coupled to 10 to give 10b in
91% yield. 1-Phenylethylamine c proved to be a poor donor
overall, but 3-phenyl-1-methylpropylamine d was coupled with 8
to form amide 8d with a conversion of 17%. Some of the -
carboline amides synthesized here have been shown to have
biological activity, most notably against benzodiazepine
receptors, first reported in the 1980s,15 and are of interest as
treatments against, for example, alcohol abuse.16
In order to investigate the broader applicability of McbA to
amide bond synthesis, aryl acid substrates 12-19, with
structures more diverged from the -carboline acid platform,
were then tested with 2-phenylethylamine a, in an effort to
produce amide products 12a to 19a (Table 2).
Table 2. ATP-dependent coupling of 12-19 with amine a by McbA. Values
represent % conversions to amide products after 24 h as determined by HPLC
analysis.
Scheme 2. -carboline carboxylic acid and amine partners for McbA-catalyzed
amide bond formations.
Acid
Product
Conversion (%)
Carboxylic acid substrates 6 and 8-11 were synthesized using
Pictet-Spengler condensations of tryptophan-derived esters with
relevant aldehydes, followed by hydrolysis of the ester products,
as detailed in the Supporting Information. Substitution of the
acyl group in position 1 of the pyridine ring with a secondary
hydroxymethyl (8) reduced the conversion to 21% of product 8a
after 1 h, but 96% conversion was achieved after 24 h. An
increase in the size of the substituent to benzoyl 9 gave
conversions to product 9a of 8% and 82% after 1 h and 24 h
respectively. Replacement by an ethyl group 10 showed that an
oxygen atom on this substituent was not necessary, with 100%
conversion to product 10a again achieved after 24 h. Even
replacement with H (11) resulted in 6% conversion to 11a after
24 h. The activity of the enzyme was then tested using
carboxylic acids 6 and 8-11 with amines a-d (Scheme 2, Table
1).
12a
13a
14a
15a
16a
17a
18a
19a
34
23
43
40
41
39
4
12
13
14
15
16
17
18
19
24
Overall McbA displayed a surprisingly broad substrate
range, including substrates such as naphthoic acid 14, indole
carboxylic acids 15 and 16 and benzofuran-5-carboxylic acid 17,
although quinoline substrate 18 was transformed with low
conversion. Strikingly, McbA, was also able to couple benzoic
acid 19 with a with a modest but significant conversion of 24%.
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10.1002/anie.201804592
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COMMUNICATION
To investigate the promiscuity in aryl acid recognition by
McbA, the structure of the enzyme was determined using X-ray
The substrate 6 is bound within a hydrophobic pocket
stacked between the loop between G295 and F301 on one face
and L202 on the other (Figure 2). Few specific interactions
between active site residues and the substrate are made, except
that the carbonyl oxygen and pyridine nitrogen of 6 interact with
the backbone N-H and C=O of G295 at distances of
approximately 3.9 and 3.4 Å respectively. The relative lack of
specific interactions may help to explain the relaxed specificity of
McbA for the range of aryl acids used in this study. The
exception was quinoline derivative 18, in which the N-atom in
the 1-position has had an adverse effect on conversion, perhaps
disrupting the hydrophobic interaction made by the native ligand
with F301. Residue D201, which points away from 6 in the
active site, is replaced in CARs and DhbE by H300, and is close
to the benzoic acid carboxylate in the CAR-AMP complex 5MSD.
This histidine has been implicated in the catalysis of both
adenylation and thiolation reactions in 4-chlorobenzoyl-CoA
ligase (4CBL, 3CW8, 3CW9).23,24
The amidation conformer of McbA superimposes well with
the thiolation conformer of 4-CBL (3CW9, 2.0 Å over 430 C
atoms) and also the ‘A’ domain in the structure of CAR-A-PCP
(5MSS, 2.5 Å over 402), the construct used in amide-bond
forming reactions described previously.9 The structure of 4-CBL
in the thiolation state, in complex with 4-chlorophenacyl- CoA (4-
CPA-CoA, 3CW9, Figure S3a)23 locates the binding site for the
thiolation product, and permits a comparison with the equivalent
region of McbA (Figure S3b) The surfaces of the enzymes show
that positively charged residues in 4-CBL, such as R87, which
interact with the phosphates in 4-CPA-CoA, are absent in McbA;
indeed the entrance to the active site features three residues
with carboxylate side chains, D463, E221 and E400 which may
favour interaction with incoming amine substrates.
crystallography.
A combination of a K483A mutant, co-
crystallised with AMP, magnesium ions and the -carboline acid
substrate 6, yielded crystals of diffraction quality. The structure
was solved using molecular replacement, and refined to a
resolution of 2.80 Å (Data collection and refinement statistics
can be found in the Supporting Information, Table S2). McbA
adopts a structure within the ANL superfamily of adenylating
enzymes defined by Gulick,17 and which includes firefly
luciferase,18 acetyl-CoA synthase,19 and standalone adenylation
domains of NRPSs, including the dihydroxybenzoate
adenylating enzyme enzyme DhbE20 from the bacillibactin
biosynthetic pathway. Each ANL enzyme is characterised by a
two-domain structure in which a large N-terminal domain is
coupled to a smaller C-terminal one, of 4-500 and 100 amino
acid residues respectively. Crucially, the structures of many ANL
enzymes have revealed substantial relative domain rotation
during the reaction coordinate, with one conformation
(‘adenylation’) assumed for adenylate formation from the
carboxylate substrate and ATP, and a second, (‘thiolation’) in
which the C-terminal domain rotates to enable the ligation
reaction between the phosphopantetheinate thiol and the
adenylate intermediate.
The McbA structure features the two predicted domains: the
large N-terminal domain of residues 1-394 (McbAN) and the
smaller C-terminal one featuring residues 395-494 (McbAC), with
the hinge residue at Q394. The McbA structure is unusual
however in that, of five molecules within the asymmetric unit,
four (A-D) are in the ‘adenylation’ conformation (Figure 1a) and
one (E) is in the ‘thiolation’ state described above, which we
have termed ‘amidation’ (Figure 1b) for McbA. The adenylation
conformer of McbA superimposes well with other ANL enzymes
crystallized in the same state, including PheA (1AMU, 2.1 Å over
431 C atoms),21 DhbE (1MD9, 2.3 Å over 445)20 and the
recently determined structure of the relevant ‘A’ domain from
CAR from Nocardia iowenis (NiCAR) in complex with benzoic
acid and AMP (5MSD, 2.8 Å over 406).22
The McbA-catalyzed synthesis of selected amides was
scaled up to utilise 50 mg (0.20 mmol) of aryl acids 6, 10, 14, 16,
17 and 19 in a 50 mL reaction volume, with 1-5 molar
equivalents 2-phenylethylamine a and two equivalents of ATP
(198 mg; 0.40 mmol). Amide products 6a, 10a, 14a, 17a and
Figure 1. Structures of McbA in the adenylation (a) and amidation (b) conformations. In each case the protein is coloured in green from residues 1-394
(McbAN) and coral from 395-494 (McbAC). AMP (yellow) is bound at the domain interface and 6 (blue) within a binding pocket in the N-terminal domain. The
McbAC domain in b is rotated 149° relative to the larger McbAN domain compared with their orientations in a.
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10.1002/anie.201804592
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HPLC analyses, biotransformation protocols, crystallisation and data
collection and refinement can be found within the Supporting Information.
Acknowledgements
M.P. received an iCASE studentship award from GSK in
collaboration with the Biotechnology and Biological Sciences
Research Council (BBSRC). A.C. thanks the Principado de
Asturias for a Clarín postdoctoral fellowship. We thank Dr Johan
P. Turkenburg and Mr Sam Hart for X-ray data collection, and
the Diamond Light Source for access to beamline i03 under
proposal number mx-9948.
Keywords: amides • biocatalysis • adenylation • ligase • ATP
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Figure 2. Active site of adenylation conformer of McbA showing residues
involved in the binding of substrate 6 and AMP. Electron density is the Fo-Fc
(omit) map at a level of 3 before inclusion of the ligand atoms in refinement.
Ligand atoms have been added for clarity. Selected interactions are indicated
by black dashed lines.
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on 50 mg scale (*carried out on 30 mg scale).
Acid
6
Equivalents of amine
Isolated Yield (%)
2
85 (6a)
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favourably with the application of CAR-A-PCP to preparative
amide bond formation,9 as 100 equivalents of amine donor were
required for those reactions, against the low ratios reported
here. The difference may reflect of the active participation of
McbA in amine binding and catalysis of amidation, which was
not yet established for the CAR-A-PCP reaction. The results
offer promise for the application of McbA-type enzymes in
preparative biocatalyzed reactions, particularly if ATP recycling
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The efficient biocatalytic formation of amide bonds for the
preparation of pharmaceutical-type amides, is one of the most
significant reactions for which biocatalytic alternatives are being
sought. The studies presented here suggest that McbA and
related ABS enzymes may provide starting points for the
evolution of activity towards a wider range of carboxylic acid and
amine partners, using the structure of McbA as a basis for
structure-guided evolution.
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Experimental Section
Details of target selection, gene cloning and expression, enzyme
purification and assay, synthesis of substrates and product standards,
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COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Mark Petchey,[a] Anibal Cuetos,[a]
Benjamin Rowlinson,[a] Stephanie
Dannevald,[a] Amina Frese,[a] Peter
W. Sutton,[b]† Sarah Lovelock,[b]‡
Richard C. Lloyd,[b] Ian J.S.
Fairlamb[a]* and Gideon Grogan[a]*
The amide bond synthetase McbA
from
Marinactinospora
thermotolerans catalyzes the ATP
dependent two-step synthesis of
phenyethylamides
using
a
surprisingly broad range of aryl
acid donors and at amine molar
equivalents of just 1 to 5. The
structure of the enzyme reveals the
basis for broad aryl acid specificity
and serves as a platform for further
engineering of this system for the
biocatalytic synthesis of amides.
Page No. – Page No.
Aryl acid specificity of the amide
bond synthetase McbA suggests
broad potential for the biocatalytic
synthesis of amides
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