An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket

Article abstract of DOI:10.1002/anie.201900318

Adenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A-domains. We show that a single asparagine-to-glycine mutation in an aryl acid A-domain leads to an enzyme that tolerates a wide range of non-native aryl acids. The engineered catalyst is capable of activating non-native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

Full text of DOI:10.1002/anie.201900318

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: An engineered aryl acid adenylation domain with a capacious
active site microenvironment
Authors: Fumihiro Ishikawa, Akimasa Miyanaga, Hinano Kitayama,
Shinya Nakamura, Isao Nakanishi, Fumitaka Kudo, Tadashi
Eguchi, and Genzoh Tanabe
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201900318
Angew. Chem. 10.1002/ange.201900318
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10.1002/anie.201900318
Angewandte Chemie International Edition
COMMUNICATION
An engineered aryl acid adenylation domain with a capacious
active site microenvironment
Fumihiro Ishikawa,+*[a] Akimasa Miyanaga,+[b] Hinano Kitayama,[a] Shinya Nakamura,[a] Isao
Nakanishi,[a] Fumitaka Kudo,[b] Tadashi Eguchi,[b] and Genzoh Tanabe[a]
Abstract: Adenylation (A) domains act as the gatekeepers of non-
ribosomal peptide synthetases (NRPSs) ensuring the activation and
thioesterification of the correct amino acid/aryl acid building blocks.
Aryl acid building blocks are most commonly observed in iron-
chelating siderophores, but are not limited to them. Very little is
known about the reprogramming of aryl acid A-domains. Here we
show that a single asparagine-to-glycine mutation in an aryl acid A-
domain creates novel enzyme specificities toward a wide range of
non-native aryl acids. The engineered catalyst is capable of
activating the non-native aryl acids functionalized with nitro, cyano,
bromo, and iodo, even though no enzymatic activity of wild-type
enzyme was observed toward these substrates. Co-crystal
structures with non-hydrolysable aryl-AMP analogues revealed the
origins of substrate promiscuity expansion, highlighting an enlarged
substrate binding pocket of the enzyme. Our finding may be
exploited to produce diversified aryl acid-containing natural products
modules.[8,9]
Aryl acids such as the common 2,3-dihydroxybenzoic acid
(DHB) and salicylic acid (Sal) of siderophores, are an important
class of NRP building blocks,[10] but not limited to them. The
unique building blocks including anthranilic acid (Ant), p-
aminobenzoic acid, and 3-hydroxypicolic acid have been
observed
in
acetylaszonalenin,[11]
albicidin,[12]
and
pyridomycin,[13] respectively. Our knowledge about the non-
ribosomal codes toward aryl acid building blocks is much more
limited. Engineering efforts focusing on aryl acid A-domains are
therefore limited so far. The recognition properties of DhbE, an
aryl acid A-domain from the siderophore bacillibactin
biosynthetic pathway, has been manipulated from DHB toward
Ant with a 206-fold specificity switch via directed evolution based
on yeast display technology.[14] The substrate specificity of a
NRPS-like protein that can activate 5-methyl orsellinic acid has
been engineered toward Ant with a 26-fold improvement in
and serve as
a template for further directed evolution in
combinatorial biosynthesis.
enzyme
specificity
via
bioinformatics
analysis
and
mutagenesis.[15] Aryl acid A-domains frequently display the
ability to catalyze adenylation toward a range of structurally
related aryl acids.[13,16] The substrate promiscuity would be
afforded by a common benzoic acid (BA) recognition moiety.
Presumably, the aryl acid A-domains form the fine-tuned active
sites to accommodate the differences in substituents between
aryl acid substrates by an appropriate cavity and/or a charge. In
this study, we show that an engineered archetypal aryl acid A-
domain (DHB-specific) exhibits a capacious active site which
exhibits novel specificity toward aryl acids containing nitro,
cyano, chloro, bromo, and iodo functionalities.
Peptide-based natural products known as non-ribosomal
peptides (NRPs) are one of the most promising resources for
drug discovery and development owing to a broad range of
biologically important properties, including antimicrobial,
anticancer, and immunosuppressive activities.[1] They are
biosynthesized by multimodular enzymes known as non-
ribosomal peptide synthetases (NRPSs).[2] Each module is
responsible for selection, activation, and incorporation of a
building block of the growing intermediate. Common to all NRPS
machinery are the peptidyl carrier protein (PCP) and adenylation
(A) domains; their activities are essential for incorporation of
amino acid building blocks into the NRPS machinery. The A-
domain activates an amino acid substrate at the expense of
adenosine triphosphate (ATP) and facilitates covalent
attachment of it to the holo-PCP at the thiol group of 4′-
phosphopantetheine (Ppant),
a cofactor post-translationally
attached to a conserved Ser on the PCP. The A-domain catalyst
serves as an entry point for an amino acid into NRPS machinery.
Therefore, the A-domain makes them attractive targets for the
engineering of NRPS machinery for the production of non-native
NRPs.[3] Many strategies have been developed, including
precursor-directed biosynthesis,[4] mutasynthesis,[5] active-site
manipulation,[6] directed evolution,[7] and swapping domains and
Scheme 1. Nonribosomal peptide biosynthesis of the siderophore
enterobactin. DHB, 2,3-dihydroxybenzoic acid; ATP, adenosine triphosphate;
PPi, pyrophosphoric acid; AMP, adenosine monophosphate; A, adenylation
domain; ArCP, aryl carrier protein; ICL, isochorismate lyase.
[a]
Dr. F. Ishikawa,[+]* H. Kitayama, Dr. S. Nakamura, Prof. I. Nakanishi,
Prof. G. Tanabe
Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-
Osaka, Osaka 577-8502, (Japan)
*E-mail: ishikawa@phar.kindai.ac.jp
EntE is part of a three-module NRPS assembly line involving
EntB, EntE and EntF; it is responsible for the biosynthesis of the
siderophore enterobactin in Escherichia coli.[17] EntE is a stand-
alone aryl acid A-domain that catalyzes the activation of DHB,
and transfers it to the ArCP domain of EntB (Scheme 1). EntE
displays the ability to catalyze adenylation toward a few number
[b]
[+]
Dr. A. Miyanaga,[+] Dr. F. Kudo, Prof. T. Eguchi
Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-
okayama, Meguro-ku, Tokyo 152-8551, (Japan)
These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for this the
author(s) of this article can be found under: XXX.
This article is protected by copyright. All rights reserved.

10.1002/anie.201900318
Angewandte Chemie International Edition
COMMUNICATION
of non-native aryl acids.[18-20] The crystal structure of EntE
complexed with the ArCP domain of EntB has been determined
with a Sal-based vinylsulfonamide inhibitor (Figure S1A).[21] The
crystal structure revealed that the hydroxyl group of the Sal-
based inhibitor is hydrogen-bonded to the Asn235 side-chain,
demonstrating an essential role for recognition of the 2-OH of
aryl acid substrates (Figure S1A). The cocrystal structure of the
homologous DhbE and DHB reveals that hydroxyl groups of
DHB are hydrogen-bonded to the Ser240O (3-OH) and
Asn235N2 (2- and 3-OH) (Figure S1B).[22] Since we intended to
open up a cavity large enough to accommodate aryl acids
bulkier than DHB and Sal, we focused on optimizing interactions
between EntE and the C-2 functionalities in the aryl acid
substrates. Replacing the Asn235 with smaller amino acids
To obtain a detailed understanding of substrate selectivity and
enzyme active-site microenvironments, we determined catalytic
parameters of the adenylation reaction catalyzed by wtEntE and
the N235G variant using 12 BA derivatives (Figures S11–S22
and Tables 1, S2, and S3). The members of group I were
categorized as BA derivatives with modifications at the 3-
position. wtEntE afforded calculated kcat/Km values of 2.1, 9.9, 14,
3.2, and 1.3 mM-1 min-1 toward 3-ethynylBA, 3-fluoroBA, 3-
chloroBA, 3-bromoBA, and 3-iodoBA, respectively. In contrast,
the kcat/Km values of the N235G variant with 3-ethynylBA, 3-
fluoroBA, 3-chloroBA, 3-bromoBA, and 3-iodoBA were 16, 24,
13, 11, and 23 mM-1 min-1, respectively. These changes
correspond to 30-, 10-, 3.8-, 14-, and 72-fold switch in substrate
specificity relative to wtEntE. The N235G variant efficiently
should overcome the limited size of the substrate binding-pocket. catalyzed the adenylation reaction of 3-nitroBA (kcat/Km = 12 mM-
1
We therefore decided to generate variants (N235G, N235A,
N235S, and N235T) (Figures S2-S6). The resulting single
variants (N235G, N235A, N235S, and N235T) were individually
tested against DHB and Sal substrates (Figures S7–S10, Table
S1, and Supplementary Discussion). The N235G variant had a
smaller effect on the kcat and Km values measured for DHB and
Sal substrates, making it the most active catalyst toward both
substrates in the mutant enzymes tested. It would be expected
that the N235G mutation affords a capacious active site that is
capable of accommodating a large variety of aryl acids with
different functionalities bulkier than hydroxyl group at the 2-
position of BA.
min-1) and 3-cyanoBA (kcat/Km = 18 mM-1 min-1), whereas
wtEntE showed no catalytic activity toward these substrates.
The members of group II were categorized as BA derivatives
with modifications at the 2-position. The kcat/Km value of the
N235G variant with the 2-fluoro substituent was 5.7 mM-1 min-1
comparable to that of wtEntE, which resulted in a nearly 5-fold
switch in substrate specificity. In contrast, the N235G variant
displayed a lower Km value for 2-chloroBA (Km = 28 ± 6 µM),
which is approximately 90-fold lower than that of wtEntE with 2-
chloroBA. Our kinetics data indicate that the N235G variant
switched the enzyme specificity 203-fold toward 2-chloroBA.
Furthermore, the N235G variant afforded significant changes in
substrate specificity toward 2-nitroBA (kcat/Km = 2.4 mM-1 min-1),
2-bromoBA (kcat/Km = 3.8 mM-1 min-1) and 2-iodoBA (kcat/Km = 2.2
mM-1 min-1) with bulky 2-position substituents, albeit with wtEntE
having no catalytic activity toward these substrates.
We next determined the substrate profiles of wtEntE and the
N235G variant toward a group of 22 structurally related BA
derivatives with different modifications at the 2- or 3-positions
using
a
nonradioactive high-throughput malachite green
colorimetric assay[23] (Figure 1 and Supplementary Discussion).
To test whether the N235G variant could interact with
downstream domains and process non-native aryl acid
substrates, we used an ArCP domain of EntB from the
enterobactin synthetase (Scheme 1). We examined the transfer
of 11 non-native aryl acids (3-nitroBA, 3-cyanoBA, 3-ethynylBA,
3-fluoroBA, 3-chloroBA, 3-bromoBA, 2-fluoroBA, 2-chloroBA, 2-
bromoBA, 2-iodoBA, and 2-nitroBA) and 2 native substrates
(DHB and Sal) to the Ppant group of the ArCP catalyzed by the
N235G variant using MALDI-TOF-MS (Figures S23–S35). A
MALDI assay showed that the N235G variant is capable of
loading ≥ 90% of the ArCP domain of EntB with the substrates
(DHB, Sal, 3-ethynylBA, 3-fluoroBA, 3-chloroBA, 2-fluoroBA, 2-
chloroBA, 2-bromoBA, 2-iodoBA, and 2-nitroBA). In contrast, the
N235G variant gave 50 to 70% loading of the ArCP domain of
EntB with 3-nitroBA, 3-cyanoBA, and 3-bromoBA. This may be
because ArCP-Ppant-aryl acid species could be easily
hydrolyzed to ArCP-Ppant given by their instability resulting from
electron-withdrawing substituents at the 3-position.
To understand the structural basis for this promising specificity,
we solved X-ray crystal structures of the N235G variant
complexed with non-hydrolysable aryl-AMP analogues (aryl-
AMSs). We therefore synthesized a set of aryl-AMSs using DHB,
Sal, 3-nitroBA, 3-cyanoBA, 2-nitroBA, 2-chloroBA, and 2-
bromoBA as ligands (Scheme S1) and subsequently constructed
inhibition profiles of wtEntE and the N235G variant (Figures
S36–S43 and Table S4). We chose 3-cyanoBA-AMS and 2-
nitroBA-AMS for co-crystallization with the N235G variant
because of tight- and specific-binding properties toward the
Figure 1. Substrate profiles of wtEntE and the N235G variant. Wild-type EntE
and the mutant EntE (N235G) were used at 1 µM with 1 mM of aryl acid
substrates described. Controls were treated identically except no substrates
were added to the reaction buffer. To estimate the relative adenylation
activities, we subtracted the A620 value of the reaction mixture without
substrate from the A620 values of reaction mixtures containing substrate.
Shown are average values from two independent experiments in duplicate. To
be considered an active substrate, OD620 values > 0.2 were required. It should
be noted that all aryl acid substrates were active at least to some extent.
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10.1002/anie.201900318
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COMMUNICATION
N235G variant, with calculated Kiapp values of 183 ± 28 nM and
50 ± 3 nM, respectively (Figure 2A and Table S4), which should
allow to provide a readout of the molecular basis for the altered
specificity.
N235G substitution enlarges the substrate binding site by ~25 Å3
compared to that of wtEntE, providing a cavity for bulky
substituents at 2- or 3-positions of BA (Figures 2B–D and S45).
To obtain detailed insights into the substrate recognition
mechanism of the N235G variant, we compared the positions of
aryl-AMS ligands in these N235G variant structures. The AMS
moieties of both aryl-AMS ligands occupy almost the same
position in the nucleoside-binding pocket of wtEntE (Figures
S46), whereas the position of the aryl acid moiety is slightly
different between these two structures (Figure S46C). The
position of the 2-nitroBA moiety in the EntE (N235G)2-nitroBA-AMS
structure is similar to that of the Sal moiety in the EntE-EntB
(ArCP) structure (Figure S46B), in which the 2-hydroxy group of
the Sal moiety orients toward the Asn235 side-chain through
formation of a hydrogen bond. The 2-nitro group of the 2-nitroBA
moiety forms hydrogen bonding interactions with the backbone
amide groups of Tyr236 and Ala335, and it orients toward the
space created by the N235G substitution in the EntE (N235G)2-
Table 1. Catalytic parameters of the adenylation reaction catalyzed by wtEntE
and the N235G variant.[a]
Vari.
Substr.
Km
kcat
[min-1
kcat/Km
[mM-1
Specificity
[b]
Specificity
switch[c]
[µM]
]
min-1
]
WT[d]
WT[d]
WT
DHB
2.0 ± 0.8
28 ± 8
n.d.[e]
2.6 ± 0.2
11 ± 0.9
n.d.[e]
1311
412
n.d.[e]
n.d.[e]
2.1
1
-
Sal
0.31
n.d.[e]
n.d.[e]
-
3-NitroBA
3-CyanoBA
3-EthynylBA
3-FluoroBA
3-ChloroBA
3-BromoBA
3-IodoBA
2-NitroBA
2-FluoroBA
2-ChloroBA
2-BromoBA
2-IodoBA
DHB
-
WT
n.d.[e]
n.d.[e]
-
WT
1027 ± 221
965 ± 129
728 ± 58
738 ± 158
1987 ± 293
n.d.[e]
2.2 ± 0.2
9.5 ± 0.6
10 ± 0.3
2.3 ± 0.2
2.6 ± 0.2
n.d.[e]
1.6×10-3
7.5×10-3
1.0×10-2
2.4×10-3
1.0×10-3
n.d.[e]
2.2×10-3
2.3×10-4
n.d.[e]
-
WT
9.9
-
WT
14
-
WT
3.2
-
structure. In contrast, the orientation of the 3-cyanoBA
nitroBA-AMS
WT
1.3
n.d.[e]
-
WT
-
moiety in the EntE (N235G)3-cyanoBA-AMS structure is relatively
different (Figure S46A). The aromatic ring of the 3-cyanoBA
moiety rotates ~20º, probably because of steric hindrance with
the side-chain of Ser240. This rotation allows the 3-cyano group
of 3-cyanoBA moiety to orient toward the space created by the
N235G substitution. Thus, the N235G substitution is sufficient to
accommodate the bulky substituents at the 2- or 3-positions of
BA.
WT
388 ± 90
2574 ± 1017
n.d.[e]
1.1 ± 0.09
0.76 ± 0.19
n.d.[e]
2.8
-
WT
0.30
n.d.[e]
n.d.[e]
318
157
12
-
WT
-
WT
n.d.[e]
n.d.[e]
n.d.[e]
-
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
NG[g]
5.2 ± 0.9
22 ± 4
1.7 ± 0.08
3.4 ± 0.2
6.3 ± 0.4
8.3 ± 0.2
1.5 ± 0.1
2.9 ± 0.2
2.2 ± 0.07
2.2 ± 0.2
1.4 ± 0.03
0.58 ± 0.03
1.5 ± 0.1
0.41 ± 0.02
0.60 ± 0.04
0.78 ± 0.04
1
-
Sal
0.49
1.6
[f]
3-NitroBA
3-CyanoBA
3-EthynylBA
3-FluoroBA
3-ChloroBA
3-BromoBA
3-IodoBA
2-NitroBA
2-FluoroBA
2-ChloroBA
2-BromoBA
2-IodoBA
544 ± 99
471 ± 36
95 ± 26
123 ± 21
176 ± 19
196 ± 61
60 ± 6
3.7×10-2
5.6×10-2
4.9×10-2
7.5×10-2
4.0×10-2
3.5×10-2
7.2×10-2
7.5×10-3
1.8×10-2
4.6×10-2
1.2×10-2
6.9×10-3
-
[f]
18
-
16
30
10
3.8
14
72
24
13
11
23
[f]
241 ± 39
265 ± 52
28 ± 6
2.4
-
5.7
8.3
15
203
[f]
160 ± 38
354 ± 60
3.8
-
[f]
2.2
-
[a] Catalytic parameters were determined by a coupled hydroxamate-MesG
continuous spectrophotometric assay.[24] For additional kinetics data and
experimental errors, see Figures S11–S22 and Tables S2 and S3. [b]
Specificity refers to the relative specificity for the indicated substrates with
respect to DHB: (kcat(substrate)/Km(substrate))/(kcat(DHB)/Km(DHB)). [c] The specificity
switch shows specificity(N235G)/specificity(wt) for the indicated aryl acid. [d]
Values taken from ref. [25]. [e] n.d.: not determinable. [f] We could not
estimate specificity switch for these substrates because of the lack of wtEntE
activities toward the corresponding substrates. [g] N235G.
The structures of EntE N235G proteins in complex with 3-
cyanoBA-AMS (EntE (N235G)3-cyanoBA-AMS, PDB 6IYL) and 2-
nitroBA-AMS (EntE (N235G)2-nitroBA-AMS, PDB 6IYK) have been
determined at 2.56 Å and 2.45 Å resolution, respectively
(Figures S44–S46 and Table S5). The overall structures of these
EntE (N235G) complexes are almost identical to the EntE
component of EntE-EntB (ArCP) complex (root mean square
deviation of 0.44–0.45 Å for C atoms of chain A). Recent
structural studies demonstrated that the multiple distinct
conformations adopted by A-domains guide transfer of
intermediates between domains in their catalytic cycles.[21,26,27]
Figure 2. Structural analysis of the N235G variant. (A) Structures of Non-
hydrolysable aryl-AMP analogues 3-cyanoBA-AMS and 2-nitroBA-AMS. (B)
The substrate-binding pocket of the EntE-EntB (ArCP) complex (PDB code:
3RG2) using a Sal-based vinylsulfonamide inhibitor.[20] The Asn235 side-chain
and Sal-based vinylsulfonamide inhibitor are shown as sticks. (C) The
substrate-binding pocket of EntE (N235G)3-cyanoBA-AMS (PDB 6IYL) complexed
with 3-cyanoBA-AMS. (D) The substrate-binding pocket of EntE (N235G)2-
The N235G variant structures show
a thioester-forming
conformation that would be adopted to catalyze the covalent
attachment of aryl acid units onto ArCP, as observed in the
structure of the EntE-EntB (ArCP) complex (Figure S44). The
(PDB 6IYK) complexed with 2-nitroBA-AMS. The 3-cyanoBA-AMS
nitroBA-AMS
and 2-nitroBA-AMS molecules are shown as sticks.
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10.1002/anie.201900318
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COMMUNICATION
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range of non-native aryl acids as a substrate and transferring
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partly supported by a Grant-in Aid for Scientific Research on
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Scientific Research (C) (17K07747 to A.M.) from the Japan
Society of the Promotion of Science (JSPS) and the Takeda
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Keywords: non-ribosomal peptide synthetase • aryl acid
adenylation domain • engineering • mutagenesis • structural
biology
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This article is protected by copyright. All rights reserved.

10.1002/anie.201900318
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
A capacious pocket: A single
mutation in an aryl acid adenylation
domain creates novel enzyme
specificities toward a wide range of
non-native aryl acids. In particular,
the engineered variant is capable of
activating the non-native aryl acids
functionalized with cyano, nitro,
bromo, and iodo, albeit with the wild-
type enzyme having no activity
toward these substrates. Co-crystal
structures with non-hydrolysable aryl-
AMP analogues revealed the origins
of the substrate promiscuity,
Fumihiro Ishikawa,* Akimasa
Miyanaga, Hinano Kitayama, Shinya
Nakamura, Isao Nakanishi, Fumitaka
Kudo, Tadashi Eguchi, Genzoh Tanabe
Page No. – Page No.
An engineered aryl acid adenylation
domain with a capacious active site
microenvironment
illuminating the enlarged active site
of the variant.
This article is protected by copyright. All rights reserved.