The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine

Article abstract of DOI:10.1074/jbc.RA119.011277

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by L-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed L-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-L-2,4-diaminobutyrate (N-γ- ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ- ADABA but not the ectoine catabolic intermediate N-α-acetyl- L-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2?resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ- ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

Full text of DOI:10.1074/jbc.RA119.011277

JBC Papers in Press. Published on January 22, 2020 as Manuscript RA119.011277
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.011277
The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic
insight into the biosynthesis of the chemical chaperone ectoine
Alexandra A. Richter1,2, Stefanie Kobus3, Laura Czech1,2, Astrid Hoeppner3,,
Jan Zarzycki4, Tobias J. Erb2,4, Lukas Lauterbach5, Jeroen S. Dickschat5,
Erhard Bremer1,2,* and Sander H.J. Smits3,6,*
1Department of Biology, Laboratory for Microbiology, Philipps-University Marburg,
D-35043 Marburg, Germany
2SYNMIKRO Research Center, Philipps-University Marburg, D-35043 Marburg, Germany
3Center for Structural Studies, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
4Max-Planck-Insitute for Terrestrial Microbiology, Department of Biochemistry and Synthetic
Metabolism, Marburg, Germany
5Kekulé-Institute for Organic Chemistry and Biochemistry, Friedrich-Wilhelms-University Bonn,
D-53121 Bonn, Germany
6Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
*co-corresponding authors
Running title: Crystal structure of L-2,4-diaminobutyrate acetyltransferase
To whom correspondence should be addressed: Sander H.J. Smits, Institute of Biochemistry, Heinrich-Heine-
University Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany. Phone: (+49)-2118-112647. Fax:
(+49)-211-8115310. E-mail: sander.smits@hhu.de
To whom correspondence should be addressed: Erhard Bremer, Department of Biology, Laboratory for
Microbiology, Philipps-University Marburg, Karl-von-Frisch Strasse 8, D-35043 Marburg, Germany. Phone:
(+49)-6421-2821529. Fax: (+49)-6421-2828979. E-mail: bremer@staff.uni-marburg.de
Keywords: microbial enzyme, acetyl-coenzyme A, acetylation, crystal structure, structure-function,
osmotic stress response, chemical chaperone, L-2,4-diaminobutyrate acetyltransferase (EctA),
structural biology, reaction mechanism
________________________
Please direct all correspondence concerning this manuscript during the reviewing, editorial and printing
process to Dr. Erhard Bremer. Philipps-University Marburg, Dept. of Biology, Laboratory for Microbiology, Karl-
von-Frisch-Str. 8, D-35032 Marburg, Germany. Phone: (+49)-6421-2821529. Fax: (+49)-6421-2828979. E-Mail:
bremer@staff.uni-marburg.de
1

Abstract
Ectoine is a solute compatible with the
Introduction
Compatible solutes are a distinct
physiologies of both prokaryotic and
eukaryotic cells and is widely synthesized by
bacteria as an osmotic stress protectant.
Because it preserves functional attributes of
proteins and macromolecular complexes, it is
considered a chemical chaperone and has
found numerous practical applications.
However, the mechanism of its biosynthesis is
incompletely understood. The second step in
ectoine biosynthesis is catalyzed by L-2,4-
diaminobutyrate acetyltransferase (EctA; EC
2.3.1.178), which transfers the acetyl group
from acetyl CoA to EctB-formed L-2,4-
diaminobutyrate (DAB), yielding N-γ-acetyl-L-
group of highly water-soluble organic
osmolytes that are compliant with the
biochemistry and physiology of both
prokaryotic and eukaryotic cells (1-3). The
function-preserving attributes of these solutes
for proteins and other cellular components (4-
9) led to their description as chemical
chaperones (10-12). Building on the special
physico-chemical characteristics of these
compounds, many members of the Bacteria,
Archaea, and Eukarya use compatible solutes
as cytoprotectants against different types of
environmental and cellular challenges (1-3,13-
15).
Compatible solutes have been widely
2,4-diaminobutyrate
(N-γ-ADABA),
the
adopted by microorganisms as osmotic stress
protectants (3,16-18). Their amassing, either
through synthesis or uptake (13), increases
the osmotic potential of the cytoplasm and
prevents a long-lasting increase in the ionic
strength of this cell compartment (19,20)
under osmotically unfavorable conditions. As
an immediate result of compatible solute
accumulation, high-osmolarity-triggered water
efflux from the cell is counteracted. This in
turn prevents drop of vital turgor to
physiologically non-sustainable values, and
averts an undue increase in molecular
crowding of the cytoplasm (19-22).
substrate of ectoine synthase (EctC). Here, we
report the biochemical and structural
characterization of the EctA enzyme from the
thermotolerant bacterium Paenibacillus lautus
(Pl). We found that (Pl)EctA forms
a
homodimer whose enzyme activity is highly
regiospecific by producing N-γ-ADABA but not
the ectoine catabolic intermediate N-α-
ADABA. High-resolution crystal structures of
(Pl)EctA (at 1.2-2.2 Å resolution) for (i) its apo
form, (ii) in complex with CoA, (iii) in complex
with DAB, (iv) in complex with both with CoA
and DAB, and (v) in the presence of the
product N-γ-ADABA were obtained. To
pinpoint residues involved in DAB binding, we
probed the structure-function relationship of
(Pl)EctA by site-directed mutagenesis.
Phylogenomics shows that EctA-type proteins
from both Bacteria and Archaea are
evolutionarily highly conserved, including
catalytically important residues. Collectively,
our biochemical and structural findings
yielded detailed insights into the catalytic core
of the EctA enzyme that laid the foundation
for unraveling its reaction mechanism.
Ectoine
[(S)-2-methyl-1,4,5,6-
tetrahydropyrimidine-4-carboxylic acid] (23)
and its derivative 5-hydroxyectoine [(4S,5S)-5-
hydroxy-2-methyl-1,4,5,6-
tetrahydropyrimidine-4-carboxylic acid] (24)
are prominent members of type of compatible
solutes used by microorganisms (3). They are
widely synthesized by Bacteria (25,26), by a
restricted number of Archaea (27), and
notably, also by a few halophilic protist (28-
30), and some microalgae (31). Synthesis of
ectoine
starts
from
L-aspartate-β-
__________________________________
semialdehyde, a central hub in bacterial
amino acid and cell wall synthesis (32-36).
Ectoine is formed by sequential reactions of L-
2

2,4-diaminobutyrate transaminase (EctB; EC
2.6.1.76), L-2,4-diaminobutyrate
able to deliver ectoines on the scale of tons
annually (40). Hence, both from the
perspective of basic science and the
biotechnological production of ectoines, a
deeper understanding of the properties of the
acetyltransferase (EctA; EC 2.3.1.178), and
ectoine synthase (EctC; EC 4.2.1.108) (34,36).
A substantial sub-group of ectoine producers
can modify ectoine to yield 5-hydroxyectoine,
a biotransformation catalyzed by the ectoine
hydroxylase (EctD; EC 1.14.11.55) (37-39). In
comparison with ectoine, 5-hydroxyectoine
often possesses enhanced, or additional,
protective attributes against various types of
cellular and environmental constraints
ectoine/5-hydroxyectoine
biosynthetic
enzymes is desirable (34,36,68). Substantial
advances in this context have recently been
made though detailed biochemical and
structural studies of EctB (69), EctC (25,70),
and EctD (71-73). In contrast, the L-2,4-
diaminobutyrate acetyltransferase (EctA), the
focus of this study, is far less well understood.
EctA catalyzes the second step in
ectoine biosynthesis (34,36,41) and belongs to
the superfamily of GCN5-related N-
acetyltransferases (GNAT) (74,75). These types
of enzymes catalyze the transfer of an acetyl
group from acetyl-coenzyme A (CoA)1 to an
amino group of a range of acceptor molecules
(74,75). In the case of EctA, L-2,4-
diaminobutyrate (DAB), the reaction product
of the EctB enzyme (69), is acetylated to yield
N-γ-acetyl-L-2,4-diaminobutyrate (N-γ-ADABA)
(34,68) (Figure 1A). This intermediate is the
substrate of the ectoine synthase EctC, which
forms the cyclic ectoine molecule through a
water elimination reaction (25,34,36).
(26,40,41).
Reflecting
the
osmostress
protective role of ectoines in microbial
physiology, their enhanced production is
typically triggered when microbial cells are
exposed to high osmolarity surroundings. This
process is largely caused by a strong up-
regulation in the transcription of the
ectABC(D) biosynthetic genes (26,40-44).
Ectoines
can
also
protect
microorganisms against the detrimental
effects of extremes in either high or low
growth temperatures (39,45-47). They can
preserve the functionality of proteins against
various types of challenges (8,9,48-51),
ameliorate
desiccation
stress
(52,53),
influence membrane fluidity and stabilize lipid
bilayers (54,55), protect DNA from damage by
ionizing radiation (56,57), enhance structural
changes in DNA (58,59), provide oxidative
stress resistance (60), and possess hydroxyl
radical scavenging activities (61). Building on
the function-preserving and anti-inflammatory
properties of ectoines, various types of
medical applications of ectoines are also
increasingly pursued (62-65).
Basic biochemical properties of EctA
enzymes from H. elongata and several
methylotrophs
(34,35,68,76).
have
However,
been
a
reported
thorough
understanding of EctA is still lacking, and in
particular, crystal structures in complex with
its substrates and/or its reaction product are
missing. To fill this gap, we report here
biochemical and structural characteristics of
EctA from the thermotolerant bacterium
Paenibacillus lautus (Pl) (77) in its apo,
substrate and co-substrate bound forms, and
a crystal structure trapping the reaction
product. Collectively, this crystallographic
Reflecting their function as chemical
chaperones, ectoines have already found a
considerable
number
of
commercial
applications (26,40,66). To satisfy the
increased demand for ectoines, an industrial
scale biotechnological production process has
been developed that uses the highly salt-
tolerant bacterium Halomonas elongata as a
natural and engineered cell factory (67); it is
analysis,
combined
with
site-directed
mutagenesis experiments, illuminate the
architecture of the active site of the EctA L-
2,4-diaminobutyrate acetyltransferase and
3

allow a proposal for its enzyme reaction
mechanism.
that the EctA proteins from H. elongata and
from the methylotrophs Methylomicrobium
alcaliphilum, Methylophaga alcalica, and
Methylophaga thalassica are also dimers in
solution (34,68). Collectively, these findings
suggest that dimer-formation is probably a
Results and discussion
Overproduction and purification of (Pl)EctA
Ectoine/5-hydroxyectoine-producing
microorganisms can populate ecological
niches with rather different biological and
physico-chemical characteristics (26,40,41).
general
feature
of
EctA-type
L-2,4-
diaminobutyrate acetyltransferases.
Analysis of the purified Strep-Tag II-
(Pl)EctA and (Pl)EctA-Strep-Tag II proteins by
SDS-PAGE revealed a significant difference in
their migration behavior (Figure 1B) that was
not expected from their almost identical
calculated theoretical molecular masses. We
therefore performed an electronspray-
ionization mass spectrometry (ESI-MS)
analysis of both recombinant proteins. The
molecular masses determined by this
technique revealed values of 20.55 kDa for the
Strep-Tag II-(Pl)EctA protein and 20.12 kDa for
the (Pl)EctA-Strep-Tag II protein, respectively.
Hence, in both cases, the experimentally
determined masses correlate with the
calculated theoretical masses of the
recombinant proteins minus 0.13 kDa. This
One
of
these
ectoine-producing
microorganisms is the P. lautus (Pl) strain
Y4.12MC10, a Gram-positive spore-forming
intestinal bacterium that was originally
isolated from the effluent of the Obsidian hot
spring in the Yellowstone National Park (USA)
(77). We explored the suitability of the
(Pl)EctA protein for biochemical and
crystallographic analysis.
For the heterologous production of
(Pl)EctA in Escherichia coli, we constructed
expression vectors using a codon optimized
version of the (Pl)ectA gene (GenBank
accession number MF327591.1). These
constructs yielded (Pl)EctA proteins with
either a N-terminal (NH2-WSHPQFEK-SG) or a
C-terminal (SA-WSHPQFEK-COOH) Strep-Tag II
peptide for their purification by affinity
chromatography. The corresponding synthetic
(Pl)ectA constructs were expressed under the
control of the plasmid-based TetR-regulated
tet promoter. Both the Strep-Tag II-(Pl)EctA
and the (Pl)EctA-Strep-Tag II proteins could be
purified to apparent homogeneity (Figure 1B).
Both versions of the (Pl)EctA protein show a
dimeric state in solution, as assessed by size
exclusion chromatography coupled to multi-
angle light scattering (SEC-MALS) (78). This is
documented for the (Pl)EctA-Strep-Tag II
protein in Figure 1C and revealed a molecular
mass of 39.73 ± 0.04 kDa. The calculated
finding
could
possibly
indicate
a
posttranslational elimination of the N-terminal
methionine residues during heterologous
production and purification of the two N-
terminal and C-terminal Strep-Tag II marked
(Pl)EctA proteins. The reason for the notable
difference in their migration behavior on 15%
SDS-polyacrylamide gels is not apparent.
Kinetic parameters of (Pl)EctA
In the ectoine biosynthesis route, N-γ-
ADABA is the expected product of the EctA-
catalyzed enzyme reaction (34,36,41). In
contrast, its isomer N-α-ADABA is formed as
an intermediate during the catabolism of
ectoine when microorganisms use this
nitrogen-rich compound as a nutrient (67,79).
Notably, N-α-ADABA, but not N-γ-ADABA,
serves as the inducer for the MocR/GabR-type
EnuR regulatory protein (80) controlling the
expression of many ectoine catabolic gene
theoretical
molecular
mass
of
the
recombinant monomers are 20.68 kDa [Strep-
Tag II-(Pl)EctA] and 20.25kDa [(Pl)EctA-Strep-
Tag II], respectively. Thus, the SEC-MALS data
suggest that the (Pl)EctA protein is a stable
dimer in solution, in line with previous reports
4

clusters (41,81). To ascertain that the (Pl)EctA
enzyme exclusively synthesizes N-γ-ADABA
from L-2,4-diaminobutyrate and acetyl-CoA
(34,36) (Figure 1A), we performed an enzyme
assay in which we subsequently benchmarked
the formed reaction product(s) against
chemically synthesized and purified N-γ-
ADABA and N-α-ADABA reference samples.
We used for this experiment an HPLC analysis
protocol allowing the separation of the two
ADABA isomers (70,82). Our analysis showed
that the (Pl)EctA enzyme indeed exclusively
synthesizes N-γ-ADABA under in vitro assay
conditions (Figure 2A). Therefore, we
conclude that the EctA L-2,4-diaminobutyrate
acetyltransferase transfers the acetyl group
from the co-substrate to the substrate in a
highly position-specific manner.
(69) and of the EctC ectoine synthase (25)
from P. lautus Y4.12MC10 as these are highly
salt-tolerant enzymes. Differences with
respect to the behavior of EctA-type enzymes
from various microorganisms towards NaCl
have already been reported. Moderate
concentrations of NaCl (0.2-0.4 M) activate
the corresponding enzymes from H. elongata
and M. alcaliphilum. However, increased NaCl
concentrations inhibit those of M. thalassica
and M. alcalica (34,68), a feature shared by
the P. lautus EctA enzyme (Supporting
information Figure S1B).
Keeping the thermo-tolerant nature of
the P. lautus Y4.12MC10 donor strain in mind
(77),
a
substantial degree of thermo-
resistance of the (Pl)EctA enzyme activity was
expected. Predictably, the activity of the
(Pl)EctA enzyme increased with increasing
temperature (Supporting information Figure
1C). However, the purified protein was not
very thermostable, as the enzyme was only
active for very short times at temperatures
higher than 40 °C. For instance, at 50° C, the
(Pl)EctA enzyme was highly active but only for
a few seconds, whereupon the activity
dropped precipitously.
For the initial enzyme assays, we
determined basic biochemical characteristics
of the recombinant (Pl)EctA-Strep-Tag II
protein, including its pH-optimum, its salt
tolerance, and its optimal temperature. The
(Pl)EctA enzyme showed a broad pH-optimum
under alkaline conditions (pH 8.5-9.5),
maintaining 75 % of its activity at pH 10
(Supporting information FigureA). The enzyme
was sensitive against extreme acidic
conditions, as less then 10 % of enzyme
activity was retained at pH 6.0. Based upon
these data, we performed all subsequent
enzyme assays at pH 7.5 (75 % EctA activity) to
prevent spontaneous hydrolysis of acetyl-CoA
under alkaline conditions.
Considering the results of these basic
enzyme assays, and taking the spontaneous
hydrolysis of acetyl-CoA under alkaline
conditions and the instability of the (Pl)EctA
protein at high temperatures into account, we
devised an enzyme assay for the (Pl)EctA
protein. It employed the following conditions:
100 mM TES buffer (pH 7.5), 4 mM acetyl-CoA
or 5 mM DAB, respectively, and a temperature
of 30 °C. The following apparent kinetic
parameters for the (Pl)EctA were determined:
(i) a Km of 0.13 ± 0.03 mM and a Vmax of 44.87
± 2.66 U mg-1 for the substrate DAB, and (ii) Km
of 2.79 ± 0.73 mM and a Vmax of 58.27 ± 6.56 U
mg-1 for acetyl-CoA (Figure 2B,C).
The tolerance of the (Pl)EctA enzyme
against increased concentrations of NaCl was
rather modest: a content of 100 mM NaCl in
the assay solution already lead to a notable
decrease of the enzyme activity (down to
75 %), and only about 12 % of the enzyme
activity remained when the NaCl content of
the reaction buffer was increased to 1.5 M
(Supporting information Figure S1B). The salt-
sensitivity of the (Pl)EctA enzyme contrasts
sharply with the enzymatic characteristics of
the EctB L-2,4-diaminobutyrate transaminase
5

Crystal structure of apo-form of EctA reveals
a homodimer
bridges (Supporting information Table S1).
Notably, the dimeric assembly of (Pl)EctA also
revealed that the side chain of Tyr38 from one
monomer penetrates into the other and vice
versa (Figure 4B). This residue is part of the
amino acid sequence 36SPYCYMLLGD45 that
forms helix α2 (Figure 3A). The aromatic side-
chain of Tyr38 points towards the binding site
of the DAB substrate (Figure 4B and see
below).
In order to gain insights into the
molecular mechanisms of the EctA enzyme,
we determined crystal structures of the apo-
form of (Pl)EctA, in the presence of the two
substrates, or its reaction product (Figure 1A).
First, we determined the crystal structure of
the apo-(Pl)EctA protein; the obtained
structure had a resolution of 2.2 Å (Table 1).
The (Pl)EctA protein adopts the classical GNAT
fold (74,75) with a mixed parallel/antiparallel,
twisted β-sheet in its center, flanked by four
α-helices. A short 310-helix (α5) is present on
the extended outward-facing loop connecting
β-strand 6 and β7 (Figure 3A, B).
The crystal structures of (Pl)EctA in complex
with the co-substrate reveals an
evolutionarily conserved CoA binding site
Next, we determined the (Pl)EctA:CoA
structure by molecular replacement using the
apo-(Pl)EctA structure as search model. This
crystal structure has a resolution of 1.5 Å
(Table 1). The CoA molecule could be
unambiguously placed in the crystal structure
and refined into a well-defined density
present in the active site of (Pl)EctA (Figure 5).
A comparison of the apo- and CoA-bound
structures of (Pl)EctA revealed no major
overall differences, as indicated by a r.m.s.d.
of only 0.4 Å over 150 Cα atoms.
To identify the structurally closest
homologs of the (Pl)EctA protein, we
performed a DALI search (83) which revealed
a variety of acetyltransferases among the top
hits. The structurally closest relative of
(Pl)EctA is the L-2,4 diaminobutyric acid
acyltransferease (EctA) from the pathogen
Bordetella parapertussis (Bp) in complex with
the substrate DAB (PDB entry 3D3S: r.m.s.d. of
1.3 Å over 158 Cα atoms). No detailed
description of this crystal structure, or a
publication describing the salient features of
the (Bp)EctA enzyme is currently available. We
discuss the position of the DAB ligand in the
3D3S crystal structure below, as it differs
substantially from that which we found in the
(Pl)EctA crystal structure.
In the (Pl)EctA:CoA complex, the CoA
molecule (Figure 5A,B) is bound in a deep cleft
formed by the loops connecting α1-α2, β4-α3,
β5-α4 and helices α3 and α4 (Figure 3),
creating
a
mainly positively charged
surrounding for the co-substrate (Figure 5A,
C). The 3´-phosphate group of the adenosine
moiety interacts with the side chains of Lys94
(at a distance of 2.9 Å) and Arg125 (at a
distance of 3.0 Å), while the side chain of
Arg89 acts as a clamp that, together with the
helix α4, sandwiches the adenine moiety. As
characteristic for GNAT superfamily enzymes,
the “P-loop”, is the signature motif for the
CoA-pyrophosphate binding site (74,75,84)
and possesses the following consensus
sequence: Gln/Arg-x-x-Gly-x-Gly/Ala. The
amino acid sequence of this region present in
the (Pl)EctA protein corresponds to residues
Further inspection of the crystal
packing, and in line with our SEC-MALS
analysis (Figure 1C), revealed the presence of
an (Pl)EctA homodimer within the asymmetric
unit (AS). Two (Pl)EctA monomers are packed
against each other mainly through helices α1,
α2, the short 310-helix α5 and strand β5 and
their corresponding loop regions. Both
monomers are rotated against each other by
about 180° (Figure 4A, B). The interface area,
as calculated with the PDBePISA online server
(https://ebi.ac.uk/pdbe/pisa/), is 1505.3 Å2
and comprises 27 hydrogen bonds or salt
Arg88-Arg89-Gln90-Gly91-Ile92-Ala93.
The
6

oxygen atoms of the pyrophosphate are
involved in hydrogen bonding with the
backbone nitrogen atoms of Arg89 (at a
distance of 2.8 Å), Gln90 (at a distance of 3.3
Å), Gly91 (at a distance of 2.9 Å), Ala 93 (at a
distance of 2.9 Å) and Lys94 (at a distance of
2.9 Å). The pantothenate unit is hydrogen
bonded by its amide O atom to the backbone
of Val83 (at a distance of 2.8 Å). The β-alanine
carbonyl oxygen interacts with the side chain
of Asn120 (2.8 Å) and the cysteamine nitrogen
with the backbone hydroxyl group of Val81 (at
a distance of 2.7 Å) (Figure 5C). Although the
overall fold of (Pl)EctA does not significantly
differ upon substrate and/or cofactor binding,
it is worth mentioning that the N-terminal
part of the loop connecting helices α1 and α2
(i.e. amino acids 29-32) folds slightly closer
towards α2 in the (Pl)EctA:CoA crystal
structure, thereby enabling the tail of the
cofactor to orient towards the substrate
binding site.
using the apo-(Pl)EctA structure as search
model. Our structural analysis shows that the
DAB substrate is located in a narrow grove
that is buried inside the enzyme (Figure 4A).
Contrary to the CoA-binding site, parts of both
monomers of (Pl)EctA build up the DAB
binding site. From monomer A, the loop
connecting α1-α2, β4, the C-terminus of β5
and C-terminal part of the loop to β7 (Figure
3), contributes to the binding pocket. In
addition, helix α2 (particularly residue Tyr38)
from monomer B complete the mainly
negatively charged substrate-binding pocket
(Figure 4B and Figure 6A). DAB interacts via
both O atoms of the carboxy group with the
side chain of Gln80 and Trp79 (at distances of
3.0 Å and 3.2 Å) and the side and main chain
atoms of Asp33 (at distances of 3.2 Å and 2.8
Å). The side chain of Asp33 in turn is
coordinated via the side chain of His155 (at a
distance of 2.8 Å). The DAB nitrogen (N) is
hydrogen bonded by Asp33 and Glu158 (at
distances of 3.2 Å and 2.7 Å), whereas the
distal N atom (ND) interacts with the
backbone of Trp79 (at a distance of 2.9 Å)
(Figure 6A).
CoA-binding sites are evolutionarily
highly conserved (75,84). We therefore
assessed whether this was also true for the
corresponding binding site in the (Pl)EctA:CoA
crystal structure. Supporting information
Figure S2A-C represents overlays of the
(Pl)EctA:CoA structure with that of the Ard1
To
consolidate
our
structural
assessments of the DAB-binding site, we
probed the importance of seven DAB-
contacting residues for (Pl)EctA enzyme
activity via site-directed mutagenesis. We
replaced each of these DAB-contacting
residues separately with an Ala residue (Table
2). In an alignment of 432 bona fide EctA
proteins (41), these seven residues are either
strictly conserved or conservatively replaced
by amino acid residues with similar properties.
This is documented in an abbreviated
alignment of 15 EctA-type proteins when
(Pl)EctA was used as the search query
(Supporting information Figure S3). For all
constructed (Pl)EctA variants, we observed no
differences in the their purification in
comparison with the wild-type protein. All
mutants exhibited a significantly reduced
enzyme activity. While the His155/Ala,
acetyltransferase
from
the
archaeon
Sulfolobus sulfataricus P2 (PDB entry 2X7B),
an acetyltransferase from the bacterium
Agrobacterium tumefaciens (PDB entry 2GE3),
and the human acetyltransferase NAA50 (PDB
entry 4X5K). This comparisons highlights that
the CoA binding site in the (Pl)EctA:CoA crystal
structure
corresponds
closely
the
architecture(s) of CoA-binding sites (75,84).
The (Pl)EctA homodimer forms the binding
sites for the DAB substrate
To further understand the catalytic
mechanism of (Pl)EctA, we determined its
crystal structure bound to its substrate DAB.
This structure has a resolution of 1.5 Å (Table
1) and was solved by molecular replacement
7

Asp33/Ala, and Trp79/Ala (Pl)EctA variants
showed strongly reduced enzyme activity
(remaining activities were 29.8 %, 8.7 % and
9.9 %, respectively), the other four (Pl)EctA
variants even exhibited only residual enzyme
activity (<2 %) (Table 2). Because Tyr38 of
monomer B protrudes into the DAB-binding-
site of monomer A (and vice versa) (Figure
4B), and is apparently crucial for (Pl)EctA
enzyme activity (Table 2), we asked if the
Tyr38/Ala substitution would fundamentally
disturbe the dimer assembly of (Pl)EctA. We
therefore carried out a SEC-MALS analysis of
the (Pl)EctA-Tyr38/Ala variant and found that
it still forms a stable dimer in solution
molecule would be located at a large distance
from the DAB molecule. Since the
crystallization condition and
a
detailed
description of the (Bp)EctA crystal structure
have not yet been published, we cannot
distinguish whether this is due to
crystallization procedures, or whether this
protein contains an active site whose
architecture is substantially different from
that of (Pl)EctA. This latter possibility seems
unlikely to us because the amino acid
sequences of bona fide EctA-type proteins are
highly conserved (41) [note: the (Pl)EctA and
(Bp)EctA proteins possess an amino acid
sequence identity of 36.4% and amino acid
sequence similarity of 49.7 %].
(Supporting
information
Figure
S4).
Collectively, our mutant studies support the
position and salient characteristics of the DAB-
binding site that we have captured in the
(Pl)EctA:DAB crystal complex (Figure 6A).
The (Pl)EctA crystal structure in complex with
CoA and DAB
We succeeded in crystallizing (Pl)EctA
Among the top hits from the DALI
in complex with both the CoA cofactor and the
DAB substrate at a resolution of 1.2 Å (Table
1). While (Pl)EctA:CoA contains only one
monomer in the ASU, the ternary complex
[(Pl)EctA:CoA:DAB] crystallized as a dimer in
the ASU. This is the functional dimer, which
was observed in all crystal structures when we
inspected the symmetry-related molecules. In
the (Pl)EctA:CoA and (Pl)EctA:CoA:DAB crystal
structures, the overall fold of the protein and
the binding of CoA was very similar, as
evidenced by the low r.m.s.d. of 0.41 Å over
131 Cα atoms between both crystal
structures. The largest difference was
observed for the tail of the CoA molecule and
the loop between helix α1 and α2. The β-
alanine-cysteamine tail of CoA is oriented
slightly outwards thereby introducing an
additional hydrogen bond that is formed by
the oxygen of the Ser31 side-chain with the
N4P atom in monomer A (at a distance of 2.7
Å) (Figure 7). Through this stable interaction,
the loop between helices α1 and α2 is shifted
towards the CoA cofactor. In chain B of the
(Pl)EctA:CoA:DAB structure, the CoA tail is
bent more outwards, such that the
search with the (Pl)EctA crystal structure as
the query, only the crystal structure of the L-
2,4-diaminobutyrate acetyltransferase (EctA)
from B. parapertussis (PDB entry 3D3S)
contains DAB. The (Pl)EctA and (Bp)EctA
crystal structures possess an r.m.s.d. of 1.3 Å
over 156 Cα atoms when the structures were
overlaid with each other (Supporting
information Figure S5). However, in the
(Bp)EctA crystal structure, the DAB-binding
site is located at the interface of the two
monomers (Supporting information Figure S5),
whereas it is deeply buried in the two
monomers of the (Pl)EctA crystal structure
that we present here (Figure 4A). DAB needs
to react with acetyl-CoA for the EctA-
mediated transfer of the acetyl group to form
the reaction products N-γ-ADABA and CoA
(Figure 1A). Hence, one would expect that the
two substrates be positioned in close
proximity in the active site. In the crystal
structures of the (Pl)EctA protein, this is
indeed the case (Figure 4A) (see below).
However, in the (Bp)EctA:DAB crystal
structure, the predicted position of the CoA
8

interactions with Asn120 and Ser31 is
weakened by an increased distance to 3.7 Å.
In the (Pl)EctA:CoA:DAB structure, the DAB
substrate is located at the same position as
found in the (Pl)EctA:DAB structure. Actually,
the DAB molecule is bound by via the same set
of interactions in both crystal structures
(Figure 6A). Hence, the (Pl)EctA:CoA:DAB
crystal structure probably represents the
transition state of the (Pl)EctA enzyme.
the substrate DAB and the co-substrate acetyl-
CoA are present in an “open” conformation
(Figure 8A). In this structure, there is a
surface-exposed extended tunnel in which
acetyl-CoA will bind and a deep cavity is
present in which DAB will be bound (and
Supplementary video 1). We do not know in
which sequence of events the (Pl)EctA protein
recognizes its two substrates acetyl-CoA and
DAB. However, the kinetic parameters of the
(Pl)EctA enzyme (Figure 2B,C) suggest that
DAB might bind prior to acetyl-CoA to the
protein. In the two secondary complexes that
we obtained [(Pl)EctA:CoA and (Pl)EctA:DAB],
the chemical groups of the two substrates
involved in the acetylation reaction point
towards each other (Figure 8B,C). In the next
crystal structure, the reaction product of EctA,
N-γ-ADABA, is captured (Figure 8D and
Supplementary video 1).
The N-γ-ADABA binding site of the (Pl)EctA
enzyme
One of the crystal structures that we
obtained (PDB entry 6SJY) contained the
reaction product of the EctA enzymes, N-γ-
ADABA (Figure 1A). This crystal structure had a
resolution of 2.2 Å (Table 1). Three monomers
of the (Pl)EctA protein are present in the ASU,
and only in monomer C was the electron
density in the substrate-binding pocket
sufficiently defined to evaluate the correct
orientation of the N-γ-ADABA molecule within
the active site (Figure 6B). N-γ-ADABA forms
hydrogen bonds with Asp33, Glu158, Gln80
and Trp79 (at distances of 3.2 Å, 2.7 Å, 3.3 Å
and 3.5 Å, respectively), whereas the carbonyl
oxygen of the acetyl group transferred from
acetyl CoA onto DAB to form N-γ-ADABA
makes a H-bond to the backbone nitrogen of
Val81 (at a distance of 3.1 Å) (Figure 6B).
Notably, the enzyme reaction product N-γ-
ADABA occupies essentially the same position
and orientation as the substrate DAB within
the active site of (Pl)EctA protein (compare
Figure 6A with 6B).
In the crystal structure of the
(Pl)EctA:DAB:CoA ternary complex (Figure 8E),
the CoA molecule is somewhat differently
orientated from the position of the CoA
molecule observed in the secondary
(Pl)EctA:CoA complex. In particular, the SH-
group of CoA points in a different direction in
these two complexes (compare Figure 8B with
Figure 8E). While keeping in mind that crystal
structures only provide a snapshot of the
various states a protein can adopt, the super-
imposable positions of the substrate DAB and
the reaction product N-γ-ADABA in the
(Pl)EctA active site (compare Figure 8C with
Figure 8D) suggest that the protein backbone
and the amino acid side chains do not move
substantially during enzyme catalysis. An
overlay of the (Pl)EctA:DAB and (Pl)EctA:CoA
crystal structures revealed a distance of <3.0 Å
between the sulfur atom of the CoA molecule
and the reactive nitrogen in the γ-position of
DAB (Figure 8F). This structural comparison
suggests that these two secondary complexes
might represent stages of the L-2,4-
diaminobutyrate acetyltransferase prior to
catalysis.
Structures of the apo-, secondary-, and
ternacy-complex of (Pl)EctA represent
different steps of the catalytic cycle
By comparing all obtained (Pl)EctA
crystal structures, crucial steps in the catalytic
cycle
of
the
L-2,4-diaminobutyrate
acetyltransferase can be visualized (Figure 8
and Supporting information video S1). In the
apo-form of the enzyme, the binding sites for
9

Since acetyl-CoA is highly reactive, we
were not able to obtain crystal structures of
archaeal phyla (41), the amino acid residues
involved in the binding of the substrate DAB
and the reaction product N-γ-ADABA are
highly conserved (Supporting information
Figure S3). Furthermore, the architecture of
the acetyl-CoA-binding site of (Pl)EctA
corresponds to an evolutionarily well-
conserved fold found in microbial and
the
(Pl)EctA:acetyl-CoA,
(Pl)EctA:acetyl-
CoA:DAB, or of the (Pl)EctA:acetyl-CoA:N-γ-
ADABA complex. Instead, in our crystal
structures, the non-reactive CoA is always
present (as it was added to the crystallization
solutions). To visualize a possible tertiary
complex in which the actual co-substrate of
EctA L-2,4-diaminobutyrate acetyltransferase,
acetyl-CoA, is captured [(Pl)EctA:acetyl-
CoA:DAB], we substituted in silico the thiol
hydrogen (-SH) of CoA with an acetyl group [-
C(O)CH3] (Figure 8G). This in silico model
visualizes how close the reactive groups of
acetyl-CoA and DAB are juxtapositioned just
before the transfer of the acetyl group to DAB
occurs (Supporting information video S1).
Release of the (Pl)EctA reaction products CoA
and N-γ-ADABA from the active site would
then restore the apo-form of the EctA enzyme
(Figure 8A).
eukaryotic
acetyltransferases
(75,84).
Collectively, these data suggest that the
crystal structures of the EctA enzyme that we
present here from P. lautus (77), can serve as
the blueprint for a structural and mechanistic
understanding
of
L-2,4-diaminobutyrate
acetyltransferases in general, enzymes
catalyzing the second step of ectoine
biosynthesis (36,41,68).
From the four enzymes required for
ectoine production (EctABC) and 5-
hydroxyectoine
(EctD)
biosynthesis
(26,34,40,41,45), the crystal structure of the L-
2,4-diaminobutyrate acetyltransferase (EctA)
now complements those of the already
reported structures of ectoine synthase EctC
(25) and ectoine hydroxylase EctD (71).
Furthermore, an in silico model of the L-2,4-
diaminobutyrate transaminase (EctB) has also
been recently established and probed by site-
directed mutagenesis (69). The seminal
discovery of ectoine by Galinski et al. (23) in
the extreme halophile Ectothiorhodospira
halochloris, and of 5-hydroxyectoine by Inbar
and Lapidot (24) in Streptomyces parvulus
occurred over 30 years ago. Now, a structure-
based view of the entire biosynthetic route of
these remarkable stress protectants is finally
at hand (Figure 9; Supporting information
video S1). Collectively, this should aid now
structure-guided attempts to improve the
catalytic efficiency or stability of individual
enzymes of the ectoine/5-hydroxyectoine
biosynthetic route to increase industrial-scale
Conclusions
The five crystal structures of the
(Pl)EctA that we present here allow to trace
and visualize the steps of the L-2,4-
diaminobutyrate acetyltransferase prior and
subsequent to enzyme catalysis (Figure 8;
Supporting information video S1). Both
monomers of the (Pl)EctA dimer are crucial for
jointly building the complete architecture of
the two active sites of the dimeric enzyme
(Figure 4B). Bona fide L-2,4-diaminobutyrate
acetyltransferases are closely related proteins
as evidenced by the considerable degree of
amino acid sequence identity (Supporting
information Figure S3). Using the (Pl)EctA
protein as the search query, it ranges between
94 % for Paenibacillus glutanolyticus DSM5162
and 25 % for Oceanobacillus iheyensis HTE831
among 432 inspected EctA proteins retrieved
from a dataset of a recent comprehensive
phylogenomic analysis of ectABC gene clusters
(41). Despite that these 432 EctA proteins
originate from ten major bacterial and two
biotechnological
production
of
these
commercially valuable chemical chaperones.
10

Experimental procedures
Chemicals
mixture was then acidified with formic acid
until a pH of 3.0 was reached. The solution
was subsequently degassed and directly
applied to preparative HPLC for purification,
using a preparative 1260 Infinity system
(Agilent Technologies, Walsbronn, Germany).
Acetyl-CoA, CoA, and other contaminants
were separated using a 100 × 21 mm Gemini®
10 µM NX-C18 110 Å column (Phenomenex,
Aschaffenburg, Germany) and a mobile phase
system comprised of 25 mM ammonium
formate (pH 4.2) and methanol. Separation
was achieved using a gradient of 5 to 22 % of
methanol over 7.5 min at a flow rate of 25 ml
min-1. Acetyl-CoA was detected using a 1260
infinity diode array detector (at 260 nm) and a
6130 Quadrupole MS system (Agilent
Technologies, Walsbronn, Germany). Fractions
containing acetyl-CoA were pooled and
lyophilized for 48 hours. The dry powder of
acetyl-CoA was freshly disolved in water
before use in enzymatic assays with the
purified (Pl)EctA enzyme. The concentration of
acetyl-CoA stock solutions were calculated
from a molar extinction coefficient (ε260nm) for
saturated acyl-CoA thioesters of 16400 M-1
cm-1 (86).
Ectoine was a kind gift from the bitop
AG (Witten, Germany). Anhydrotetracycline
hydrochloride (AHT), desthiobiotin, and Strep-
Tactin Superflow chromatography material for
the purification of proteins fused to a Strep-
tag II affinity peptide were purchased from
IBA GmbH (Göttingen, Germany). The reaction
product
of
the
L-2,4-diaminobutyrate
acetyltransferase (EctA), N-γ-acetyl-L-2,4-
diaminobutyric acid (N-γ-ADABA), was
synthesized through alkaline hydrolysis of
ectoine (82). It was purified from the by-
product N-α-acetyl-L-2,4-diaminobutyric acid
(N-α-ADABA) by repeated chromatography on
a silica gel column (Merck silica gel 60) using a
gradient of ethanol/25% ammonia/water
50:1:2 – 10:1:2 as eluent (70). The identity and
purity of N-γ-ADABA was monitored by thin-
layer chromatography and nuclear magnetic
resonance spectroscopy (1H-NMR and 13C-
NMR) on a Bruker AVIII-400 or DRX-500 NMR
spectrometer
as
described
previously
(36,70,82). All chemicals used to purify N-γ-
ADABA were purchased either from Sigma
Aldrich (Steinheim, Germany) or Acros (Geel,
Belgium). Coenzyme A (CoA) tri-lithium salt
was purchased from Roche Diagnostics
(Mannheim, Germany). Other chemicals were
obtained from Sigma-Aldrich (Taufkirchen,
Germany) and Roth (Karlsruhe, Germany).
Bacterial strains, media, and growth
conditions
The E coli strain TOP10 (Invitrogen,
Carlsbad, CA, USA) was used for the
propagation of plasmids carrying ectA genes.
Cultures of the plasmid-carrying E. coli strain
were grown at 37 °C in Luria-Bertani (LB)
liquid medium (87) containing ampicillin (100
µg ml-1). Heterologous overproduction of
plasmid-encoded P. lautus EctA proteins
[(Pl)EctA] carrying a Strep-tag II affinity
peptide either at the N- or C-terminus was
carried out in the E. coli B strain BL21 in
modified minimal medium A (MMA) (87)
containing 0.5% (w/v) glucose as the carbon
source and 0.5% (w/v) casamino acids, 1 mM
MgSO4, and 3 mM thiamine as supplements.
Acetyl-CoA synthesis and purification
Acetyl-CoA was synthesized from
acetic anhydride (85) using a slightly modified
protocol. CoA (320 mg) was dissolved in 0.5 M
(8 ml) sodium bicarbonate - HCl buffer (pH
7.4). The solution was cooled down to 4 °C
and acetic anhydride (80 µl) was added drop
wise under stirring. The reaction mixture was
stirred for 30 min and the completion of the
reaction was confirmed by use of
dithionitrobenzoic acid (DTNB) detecting the
remaining free thiol groups. The reaction
11

Recombinant
construction of plasmids
DNA
procedures
and
mutagenesis using the Q5 Site-Directed
Mutagenesis Kit (New England BioLabs GmbH,
Frankfurt a. M., Germany) with custom
synthesized DNA primers purchased from
Microsynth AG (Lindau, Germany). The DNA
sequence of the entire coding region of each
mutant (Pl)ectA gene was determined by
Eurofins MWG (Ebersberg, Germany) to
ensure the presence of the desired mutation
and the absence of unwanted alterations. The
following (Pl)ectA variants were constructed:
The DNA sequence of the ectA gene
was retrieved from the genome sequence of
P. lautus strain Y412MC10 (accession number:
NC_013406.1) (77) and was used as a
template for the synthesis of a codon-
optimized version of the gene for its
heterologous expression in E. coli. Synthesis of
the (Pl)ectA gene was conducted by Invitrogen
GeneArt (Thermo Fisher Scientific, Waltham,
USA), and its DNA sequence was deposited in
the GenBank database under accession
pAR9
(GAT/GCG;
Asp33/Ala),
pAR10
(TAT/GCG; Tyr38/Ala), pAR11 (TGG/GCG;
Trp79/Ala), pAR12 (CAG/GCG; Gln80/Ala),
pAR13 (ACC/GCG; Thr115/Ala), pAR14
(CAT/GCG; His155/Ala), and pAR15 (GAA/GCG;
Glu158/Ala).
number MF327591.1. To
allow
the
overproduction and affinity purification of the
recombinant (Pl)EctA protein in E. coli, we
genetically constructed C- and N-terminal
fusions of the ectA coding region to DNA
segments encoding a Strep-tag II affinity
peptide. For this purpose, the ectA gene was
amplified from the plasmid (pLC46) provided
by the supplier of the synthetic (Pl)ectA gene
constructs using custom-synthesized primers
(Supporting information Table S2). The
resulting PCR fragment was inserted into a
pENTRY vector (IBA Göttingen, Germany),
resulting in plasmid pLC48. By applying
Stargate combinatorial cloning technology,
the ectA-coding region was then inserted into
the expression plasmids pASG-IBA3 and pASG-
IBA5 (IBA, Göttingen, Germany), respectively;
the resulting EctA overproduction plasmids
were pLC50 (EctA with N-terminal Strep-tag II;
NH2-WSHPQFEK-SG) and pLC51 (EctA with C-
terminal Strep-tag II; SA-WSHPQFEK-COOH). In
these plasmids, expression of the recombinant
(Pl)ectA gene is mediated by the tet promoter
whose transcriptional activity is regulated
through TetR, an AHT-responsive repressor
protein (IBA GmbH, Göttingen, Germany).
Overproduction,
purification,
and
determination of the quaternary assembly of
EctA proteins
Cells of the E. coli B strain BL21
harboring an (Pl)ectA expression plasmid
(either pLC50 or pLC51) were inoculated into
modified MMA containing 100 µg ml-1
ampicillin (1 L medium in a 2 L Erlenmeyer
flask) to an OD578 of 0.1 from an overnight pre-
culture prepared in LB medium. The cells were
grown on an aerial shaker (set to 180 rpm) at
37 °C until the cultures reached an OD578 of
0.5. At this time point the synthetic inducer
AHT of the TetR repressor was added to a final
concentration of 0.2 mg ml-1 to trigger
enhanced transcriptional activity of the tet
promoter and thereby boost the expression of
the plasmid-encoded (Pl)ectA gene. After 2 h
of further growth of the culture, the E. coli B
strain BL21 cells were harvested by
centrifugation (2 360 x g, at 4° C for 15 min),
re-suspended in 10 ml of purification buffer
[100 mM Tris-HCl (pH 7.5) and 150 mM NaCl],
and disrupted by passing them through a
French Pressure cell (16 000 PSI). A cleared
cell lysate of the disrupted cell was prepared
by centrifugation (31 870 x g, at 4 °C for 45
min). The cleared cell extracts of the (Pl)EctA
Site-directed mutagenesis of the (Pl)ectA
gene
Mutant derivatives of the codon
optimized (Pl)ectA gene present on plasmid
pLC51 were constructed by targeted
12

overproducing cultures was used to purify the
recombinant Strep-tag II-marked proteins by
affinity chromatography on Strep-Tactin
affinity resin as detailed previously (88,89).
The concentration of the (Pl)EctA protein in
the individual fractions eluted from the Strep-
Tactin Superflow affinity column was
To analyze the quaternary assembly of
the (Pl)EctA protein, we used size exclusion
chromatography coupled to multi-angle light
scattering detection (SEC-MALS). For these
experiments, an Agilent Technologies system
connected to a triple-angle light scattering
detector
Technology
(miniDAWN
Europe
TREOS,
GmbH, Dernbach,
Wyatt
measured
Photospectrometer
with
a
Nanodrop
(Peqlab,
ND1000
Germany) followed by a differential refractive
index detection system (Wyatt Technology)
was used. Typically, 200 μl of purified (Pl)EctA
protein (2 mg ml-1) was loaded onto the Bio
SEC-5 HPLC column and the obtained data
were analyzed with the ASTRA software
package (Wyatt Technology).
Erlangen, Germany) (25 440 M-1 cm-1). The
purity and apparent molecular mass of the
(Pl)EctA protein was assessed by SDS–PAGE
(15% polyacrylamide); the PageRuler Pre-
stained Protein Ladder (Thermo Fisher
Scientific) was used as a reference to monitor
the electrophoretic mobility of the (Pl)EctA
protein. Purified (Pl)EctA protein preparations
were concentrated to approximately 10 mg
ml-1 with Vivaspin 6 columns (Sartorius Stedim
Biotech, Göttingen, Germany) with a 10-kDa
molecular-weight cutoff value prior to
crystallization trials.
EctA enzyme activity assays
To determine the precise reaction
product of the (Pl)EctA enzyme, in other
words whether N-γ-ADABA, or N-α-ADABA (or
both) are synthesized, we carried out enzyme
assays in 20 μl 100 mM TES - HCl buffer (pH
7.5) containing 2 mM acetyl-CoA, 2 mM DAB
and 1 µg purified enzyme at a temperature of
30 °C. The reaction was stopped after 5
minutes by the addition of 20 µl acetonitrile.
The enzyme reaction product(s) were then
derivatized with Fluorenylmethyloxycarbonyl
chloride (FMOC-Cl) using a procedure based
on a previously published method (82). In
brief: 2 μl of the (Pl)EctA enzyme reaction
sample was mixed with 3 μl of an FMOC
solution (25 mg ml-1 FMOC in acetonitrile) in a
thermomixer (1 min, 900 rpm, at 20 °C).
Subsequently, the FMOC reagent that had not
chemically reacted was quenched by adding
6 μl 1-aminoadamantane (ADAM) solution
(7.6 mg ml-1 ADAM and 50 % acetone in 0.5 M
sodium borate buffer, pH 7.7) and mixed
(1 min, 900 rpm, 20 °C). The entire solution
was then diluted with 988 μl H2O, and
centrifuged (20 800 x g, at room temperature
for 10 min) to remove the denatured (Pl)EctA
enzyme. 37.5 μl of the supernatant was
injected into an HPLC system (1260 Infinity;
Agilent Technologies, Walsbronn, Germany)
The molecular mass of (Pl)EctA
proteins carrying a Strep-tag II affinity protein
attached either to its N- or C-terminus was
determined my Mass-spec analysis. 1-10 μl of
a 25 mM protein solution (in purification
buffer), were prepared by desalting the
protein solution with a MassPrep column
(Waters; Milford, USA) in a Waters ACQUITY
H-Class HPLC-system. Protein elution into the
ESI source of
a
Synapt G2Si mass
spectrometer (Waters) was performed at
60 °C with a flow rate of 0.1 ml min-1 using
isocratic elution with 5 % A (water/0.05 %
formic acid) for two minutes, followed by a
linear gradient to 95 % B (acetonitrile/0.045%
formic acid) within eight minutes and a final
holding of 95% B for four minutes. The range
of detected positive ions was 500-5000 m/z.
For
automatic
drift
correction
Glu-
Fibrinopeptide B was measured every 45s. De-
convolution of averaged spectra was
performed after baseline subtraction and
smoothing using MassLynx instrument
software with MaxEnt1 extension.
13

equipped with a 150 × 4.6 mm Gemini® 5µM
C18 110 column (Phenomenex,
protein. The pH-values of these buffer
solutions and the resulting mixtures were
adjusted with 37 % HCl or 5 M NaOH at 30 °C.
The enzyme reaction of the (Pl)EctA protein,
was started by the addition of acetyl-CoA, was
run for 100 sec and was then stopped by the
addition of 50 µl 80 % acetonitrile. To remove
denatured (Pl)EctA protein, the samples were
centrifuged (20 800 x g, at 4 °C for 5 minutes).
For the reconstitution of a neutral pH value
for the DTNB reaction, 50 µl of the
supernatant was added to 150 µl DTNB-
solution (0.2 M TES (pH 7.5), 2 mM DTNB) and
the DTNB absorption was measured using a
Tecan plate reader (Tecan Group Ltd,
Männedorf, Switzerland) at 30 °C.
Å
Aschaffenburg, Germany) and a fluorescence
detecting module (Agilent Technologies,
Walsbronn, Germany). The fluorescence-
detecting module was set to an excitation
wavelength of 266 nm and an emission
wavelength of 305 nm. The mobile phase
consisted of solvent A: 20 % acetonitrile and
0.5 % tetrahydrofuran in 50 mM sodium
acetate buffer (pH 4.2) and solvent B: 80 %
acetonitrile in sodium acetate buffer (pH 4.2).
To allow the separation of the FMOC-modified
N-γ-ADABA or N-α-ADABA isomers, the flow
rate was set to 1 ml min-1 at 40 °C using a
gradient of solvent A and B similar to the
procedure described by Kunte et al. (82).
The kinetic parameters of the (Pl)EctA
enzyme were determined using the
To determine the basic parameters of
continuous
spectrophotometric
assay
the (Pl)EctA enzyme, activity was measured at
30° C using a continuous spectrophotometric
assay. In this assay, formation of free CoA was
followed using DTNB. An extinction coefficient
(ε412nm) for DTNB of 14,000 M-1 cm-1 was used
to determine the amount of CoA released
during the enzyme reaction. The reaction
mixture (300 µl) contained 100 mM TES - HCl
buffer (pH 7.5), 1 mM DTNB, 1.2 mM acetyl-
CoA, 5 mM DAB, and 0.1 µg purified (Pl)EctA
enzyme. The enzyme reaction was started by
the addition of DAB to the reaction vessel and
was run for 1.5 min, in which the absorption
was determined. All (Pl)EctA assays were
performed using two independently produced
and purified protein preparations and each
protein sample was assayed twice. During the
screening for the temperature profile of the
(Pl)EctA enzyme, the reaction was started by
the addition of the enzyme and was run, for
high temperatures (45 – 60 °C), only for 0.3
min, due to reduced enzyme stability. The
screening for pH-optimum was performed in a
50 μl reaction volume containing a buffer
mixture [MES (pH 5.5), PIPES (pH 6.5), TES (pH
7.5), CHES (pH 7.9), HEPES (pH 8.5), and CAPS
(pH 10.0)] with 50 mM each, 1.2 mM acetyl-
CoA, 1.7 mM DAB, and 0.1 µg purified (Pl)EctA
described above with either 5 mM DAB and
varied concentrations of acetyl-CoA (0.05 –
8 mM), or with 4 mM acetyl-CoA and varied
concentrations of DAB (0.05 – 1.6 mM). The
enzyme activity of the various (Pl)EctA
mutants was monitored with the same
continuous assay in a reaction containing
100 mM TES - HCl buffer (pH 7.5), 1 mM
DTNB, 2 mM acetyl-CoA, 5 mM DAB, and
0.1 µg purified (Pl)EctA enzyme. The enzyme
activities of the (Pl)EctA variants were
benchmarked against the wild-type protein
whose activity was set to 100%. Under these
conditions the wild-type (Pl)EctA enzyme had
an activity of 29.08 +/- 4.08 U mg-1protein.
One unit is defined as the enzymatic
conversion of one µM acetyl-CoA to one µM
free CoA min-1 correlating with the same
amount of DAB converted by the (Pl)EctA
enzyme.
In silico analysis of EctA-type proteins
In a recent phylogenomic analysis of
the distribution of ect biosynthetic gene
clusters present in Bacteria and Archaea, a
curated non-redundant data-set comprising
ectoine biosynthetic genes from 437 microbial
14

species/strains was generated (41). We relied
on this dataset to retrieve EctA-type proteins
and compared their amino acid sequences
with the MAFFT multiple amino acid sequence
Apart from the apo-form different
ligand-bound complex crystals were obtained
by adding either 5 mM CoA (Coenzym A,
Sigma Aldrich), 20 mM DAB (2,4-
diaminobutyrate, Sigma Aldrich), 20 mM N-γ-
alignment
tool
(http://mafft.cbrc.jp/alignment/server/) (90)
using the (Pl)EctA protein sequence (accession
number: AWH98098) as the template for a
BLAST search (91).
ADABA
(N-γ-acetyl-2,4-diaminobutyrate
(25,82)), or the combination of CoA and DAB.
The different substrates were pre-incubated
with the protein for at least 30 min on ice. If
two substrates were used, the first one was
incubated for 5 minutes before the second
one was added. For cryoprotection, all crystal-
containing drops were overlaid with mineral
oil before the crystals were harvested and
flash frozen in liquid nitrogen.
Crystallization of the (Pl)EctA protein
Several crystals were found for the
apo-(Pl)EctA protein and the ligand-bound
forms using commercial screens (Nextal,
Qiagen,
Hilden,
Germany;
Molecular
Dimensions, Suffolk, UK) in 96-well sitting
drop plates (MRC3, Swissci) at 12 °C. Both the
C-terminal and N-terminal Strep-tag II-marked
forms of the (Pl)EctA protein were used in
these crystallization trials. Crystals of the apo-
(Pl)EctA protein were obtained using
commercial screens and by slightly optimizing
the composition of the crystallization solution.
0.1 µl (Pl)EctA protein solution (10 to 15 mg
protein ml-1) and 0.1 µl reservoir solution was
mixed and equilibrated against 40 µl reservoir
solution. For the apo-form of (Pl)EctA, the first
crystal appeared after 12 hours. The most
promising condition was found with a solution
containing 0.2 M lithium sulfate, 0.2 M sodium
acetate, 0.1 M HEPES (pH 7.5) and 25 % (w/v)
PEG 4000 from the Nextal PEG II suite (Qiagen,
Hilden, Germany) after 8 days. Best diffracting
crystals were grown in a solution consisting of
0.2-0.3 M lithium sulfate, 0.2 M sodium
acetate, 0.1 M HEPES pH 7.5 and 22-28 %
Data collection, processing and structure
determination
For the crystallographic analysis of
apo-(Pl)EctA: crystals of the ligand-free form
of (Pl)EctA diffracted to a maximum of 2.2 Å.
The dataset was collected at ID30B (ESRF,
Grenoble, France) at 100 K, processed with
XDS (92,93) and phased using the automated
AUTORICKSHAW pipeline (http://www.embl-
hamburg.de/Auto-Rickshaw/) with only the
(Pl)EctA protein sequence as input. The
resulting initial model was subsequently auto-
built using the ARPWARP webservice
(https://arpwarp.embl-hamburg.de).
After
several rounds of model building using COOT
(94) and subsequent refinement using
refmac5 (95) from the ccp4 suite (96), the
structure of the full-length apo(Pl)EctA protein
was modeled into the electron density.
The following procedures were used
(w/v) PEG 4000.
A
second condition
for the crystallographic analysis of the various
forms of the (Pl)EctA protein. For the
crystallographic analysis of (Pl)EctA:CoA: a
high resolution data set up to 1.5 Å was
collected at ID29 (ESRF, Grenoble, France) at
100 K, processed with XDS, and phased via
molecular replacement using the apo-(Pl)EctA
structure as search model. For the
crystallographic analysis of (Pl)EctA:DAB: A
high resolution data set up to 1.5 Å was
containing 0.25 M sodium sulfate, 0.1 M Bis
Tris propane (pH 8.5), 25 % PEG 3350 was also
optimized by grid screening. 1 µl (Pl)EctA
protein solution was mixed with 1 µl reservoir
solution and equilibrated against 300 µl
reservoir solution. Crystals reached their
maximum dimensions of about 100 × 200 × 50
µm3 within 5 - 13 days.
15

collected at ID30A-3 (ESRF, Grenoble, France)
at 100 K, processed with XDS, and phased via
molecular replacement using the (Pl)EctA:CoA
structure as search model. For the
crystallographic analysis of (Pl)EctA:CoA:DAB:
a high resolution data set up to 1.2 Å was
collected at ID29 (ESRF, Grenoble, France) at
100 K, processed with XDS, and phased via
molecular replacement using the (Pl)EctA:CoA
structure as search model. For the
crystallographic analysis of (Pl)EctA:N-γ-
ADABA: a data set up to 2.2 Å was collected at
ID23-1 (ESRF, Grenoble, France) at 100 K,
processed with XDS, and phased via molecular
replacement using the (Pl)EctA:CoA structure
as search model. Refinements of all complex
structures were performed as described
above.
model content of these different (Pl)EctA
crystal structures is given in Table 1. The
crystal parameters, especially the unit cell
dimensions and space group, differ between
the crystals of apo-(Pl)EctA and the crystal
with the substrates (Table 1), which implies
that a different number of (Pl)EctA proteins
are present in the asymmetric unit. In the
asymmetric unit of apo-(Pl)EctA and
(Pl)EctA:ADABA three copies of EctA were
found, whereas in (Pl)EctA:CoA and
(Pl)EctA:DAB only one monomer of the EctA
protein was present, and in the crystals of
(Pl)EctA:CoA:DAB and (Pl)EctA:ADABA two
EctA monomers were found.
PDB accession codes
The crystallographic data of the five
(Pl)EctA structures reported here have been
deposited in the RCSB Protein Data Bank
(https://www.rcsb.org/) under accession
Adding the substrate or the ortholog
of acetyl-CoA, CoA, prior to the crystallization
improved the crystal quality significantly, as
reflected by the higher resolution of the
obtained data sets. The (Pl)EctA:CoA crystal
structure was solved at 1.5 Å (Rwork and Rfree
values were 14.7% and 18.3%, respectively),
the (Pl)EctA:DAB crystal structure at 1.5 Å
(Rwork and Rfree values were 17.8% and 12.2%,
respectively), the (Pl)EctA:CoA::DAB crystal
structure at 1.2 Å (Rwork and Rfree values were
13.3% and 15.0%, respectively), and finally,
the (Pl)EctA:ADABA crystal structure at 2.2 Å
(Rwork and Rfree values were 16.7% and 20.1%,
number
[(Pl)EctA:CoA], 6SL8 [(Pl)EctA:DAB], 6SJY
[(Pl)EctA: N-γ-ADABA], and 6SLL
[(Pl)EctA:CoA:DAB], respectively.
6SLK
[apo-(Pl)EctA],
6SK1
Figure preparation of crystal structures
Figures of the crystal structures of the
(Pl)EctA protein were prepared using the
PyMol software suite (www.pymol.org) (97)
and Chimera (98).
respectively).
A
summary of the data
collection statistics, refinement details, and
16

Acknowledgements - We cordially acknowledge the staff of beamline P13 (DESY, EMBL, Hamburg,
Germany) for their kind and helpful support during crystal screening and we are equally thankful to
the staff of the ID23-1, ID29, ID30B, and ID30A-3 beamlines of the European Synchrotron Radiation
Facility (Grenoble, France) for expertly providing synchrotron radiation facilities. We greatly
appreciate the generous gift of ectoine by the bitop AG (Witten, Germany) for our study. We thank
Jochen Sohn for his excellent technical assistance during EctA protein overproduction and
purification. We are thankful to Dr. Uwe Linne (Dept. of Chemistry, Philipps-University Marburg) for
performing the mass-spec analysis of recombinant (Pl)EctA proteins; access to the Mass-spec
facilities of the Dept. of Chemistry of the Philipps-University Marburg is gratefully acknowledged. We
thank the Deutsche Forschungsgemeinschaft (DFG) for co-financing the SynaptG2Si mass
spectrometer (grant: INST 160/621-1 FUGG) to U. Linne and G. Bange). We appreciate the kind
support of Lutz Schmitt (Institute of Biochemistry, University Düsseldorf) for this project. We are very
grateful to Gert Bange for critical reading of the manuscript and many inspiring discussions. We
particular acknowledge and thank Alexander Lepak (SYNMIKRO Research Center; Philipps-University)
for producing the video visualizing the predicted enzyme reaction mechanism of the L-2,4-
diaminobutyrate acetyltransferase EctA.
This work was financially supported in part by grants from the DFG through the SFB 987
(Philipps-University Marburg; to E.B. and T.J.E.) and the DFG-funded Transregio SFB TR41
(Braunschweig/Oldenburg/Bonn/Göttingen; to J.S.D.). The Center for Structural studies is funded by
the DFG as well (grant number 417919780, INST 208/740-1 FUGG). The work of L.C. was supported
by a Ph.D. fellowship from the International Max Planck Research School for Environmental, Cellular
and Molecular Microbiology (IMPRS-Mic, Marburg).
Conflict of interest - The authors declare that they have no conflicts of interests with the content of
this article.
Author contributions - E.B. and S.H.J.S. conceived and coordinated the study and evaluated all data.
A.A.R. performed overexpression, purification, functional characterization of the EctA enzyme, and
analyzed biochemical and structural data. J.Z. and T.J.E. supported A.A.R. in designing the EctA
enzyme assay. J.Z. synthesized and purified acetyl-coenzyme A. L.C. constructed the ectA
overexpression plasmid and performed bioinformatics analysis of the phylogenetic distribution of
EctA and EctC-type proteins. S.K., A.H., and S.H.J.S. designed and performed crystallization
experiments, solved the crystal structures of EctA proteins and analyzed the data. L.L. and J.S.D.
synthesized N-γ-ADABA and analyzed its identity and purity. A.A.R., S.H.J.S, and E.B. wrote the
manuscript with input from other authors.
17

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23

1Footnotes
The abbreviations used are: CoA, coenzyme A; DAB, L-2,4-diaminobutyrate; N-γ-ADABA, N-γ-acetyl-L-
2,4-diaminobutyrate; N-α-ADABA, N-α-acetyl-L-2,4-diaminobutyrate; ESI-MS, electronspray-
ionization mass spectrometry; HPLC, high-performance liquid chromatography; ASU, asymmetric
unit; AHT, anhydrotetracycline; MMA, minimal medium A; LB, Luria-Betrani medium; FMOC,
fluoenylmethyloxycarbonyl; ADAM: aminoadamate; DTNB, dithionitrobenzoic acid.
24

Table 1. Data collection and refinement statistics for the apo form of EctA and the
substrate bound forms. Values in parentheses are for the outer shell.
Apo-EctA
CoA-EctA
DAB-CoA-EctA DAB-EctA
N-ɣ-Adaba
Crystal
parameters
X-ray source
ID30B, ESRF,
Grenoble
ID29, ESRF,
Grenoble
ID29, ESRF,
Grenoble
ID30A-3,
ESRF,
ID23-1, ESRF,
Grenoble
Grenoble
EIGER_4M
P 43 21 2
Detector
Space group
Pilatus3_6M
P 41 21 2
Pilatus_6M_F
P 43 21 2
Pilatus_6M_F
R 3
Pilatus_6M_F
P 41 21 2
Unit cell
parameters
a, b, c (Å)
176.01 176.01 68.27, 68.27,
151.21, 151.21, 57.98, 57.98,
174.22, 174.22,
60.99
61.78
79.79
46.22
134.46
α, β, ɣ (°)
90, 90, 90
90, 90, 90
90, 90, 120
90, 90, 90
90, 90, 90
Data collection
and processing
Resolution (Å)
51.89- 1.13
(1.21- 1.13)
75.53- 1.05
(1.11- 1.05)
53.24- 1.51
(1.60- 1.51)
123.2 - 2.02
(2.15 - 2.02)
124.5 - 1.95
(2.07 - 1.95)
Unique
reflections
70540
(10980)
70942 (12931) 182408
(27168)
36817
(5756)
61414 (9533)
Completness (%) 99.2 (95.5)
99.9 (99.8)
8.3 (7.8)
13.46 (1.23)
0.067 (1.343)
99.2 (96.3)
3.59 (3.19)
10.43 (1.59)
0.048 (0.608)
99.9 (99.9)
8.8 (8.8)
16.64 (1.32)
99.5 (97.2)
13.1 (12.4)
17.44 (2.45)
Redundancy
I /σ
12.9 (13.1)
10.65 (1.08)
0.161 (2.588)
Rsym
0.058 (1.453) 0.092 (1.105)
Refinement
statistics
Resolution (Å)
Rwork (%)
124.5 - 2.20
0.1695
51.883 - 1.50
0.1468 (0.143)
75.53 - 1.20
0.1218
53.40 - 1.53
0.1775
123.2 - 2.20
0.1674
(0.2370)
(0.2560)
(0.3100)
(0.2250)
Rfree (%)
0.1834 (0.185)
0.1496
(0.2680)
0.2140
(0.3220)
0.2070
(0.2700)
0.2050
(0.2690)
r.m.s.d. from
ideal
Bond lengths (Å) 0.020
0.028
2.597
15.0
0.039
3.024
18.0
0.024
2.345
32.00
0.021
2.145
44.0
Bond angles (°)
Average B-
1.983
43.0
factors (Å2)
Ramachandran
Plot
Most favoured
(%)
97.4
99.3
97.9
97.4
97.8
Allowed (%)
Disallowed (%)
Model content
Monomers/ASU
Protein residues
2.6
0.0
0.7
0.0
2.1
0.0
2.6
0.0
2.0
0.2
3
1
2
1
3
7-168, 2-176,
7- 168
-
8-167
6-169, 6-171
8-168
4-170, 2-176 ,
7-168
ADABA
220
Ligands
Water molecules 281
COA
209
COA, DAB
543
DAB
146
PDB code
6SLK
6SK1
6SLL
6SL8
6SJY
25

Table 2 Enzyme activities of the EctA variants relative to the activity of the wild-type (Pl)EctA
enzyme.
EctA variant
EctA wild-typea
Asp33/Ala
Tyr38/Ala
relative enzyme activity (%)
100
8.7 ± 3.0
0.8 ± 1.0
9.9 ± 1.5
0.8 ± 1.3
1.6 ± 1.9
29.8 ± 6.4
0 ± 0
Trp29/Ala
Gln80/Ala
Thr115/Ala
His155/Ala
Glu158/Ala
aThe enzyme activity of the (Pl)EctA wild-type enzyme was 29.08 ± 4.08 U mg-1 and was set for
comparative reasons to 100 %. Enzyme assays were conducted for the wild-type (Pl)EctA, and each of
its mutant derivatives, with two independently prepared protein batches, and in each case with two
technical replicates.
26

Figure 1. (Pl)EctA catalyzed enzyme reaction, purification, and quaternary assembly of the (Pl)EctA
protein. A, (Pl)EctA enzyme catalyzes the transfer of the acetyl-moiety from acetyl-CoA onto the
substrate L-2,4-diaminobutyrate resulting in the formation of N-γ-acetyl-L-2,4-diaminobutyrate and
free CoA as reaction products. B, Coomassie-stained SDS-PAGE of purified Strep-tag II-(Pl)EctA
(pLC50) protein and (Pl)EctA-Strep-tag II protein (pLC51) (2 µg of each protein were loaded onto the
SDS-gel). The size standard is given in kilo Dalton (kDa). (C) MALS-RI analysis shows that the (Pl)EctA
protein elutes with an absolute molecular mass of 39.73 ± 0.04 kDa consistent with the notion that
it is homodimer in solution. The calculated theoretical molecular mass of the monomer of the Strep-
tag II-(Pl)EctA and of the (Pl)EctA-Strep-tag II recombinant proteins is 20.68 kDa and 20.25 kDa,
respectively.
27

Figure 2. (Pl)EctA-dependent N-γ-acetyl-L-2,4-diaminobutyrate production and kinetic parameters
of the (Pl)EctA enzyme. A, HPLC traces showing the regio-selective acetylation of L-2,4-
diaminobutyrate (DAB) by (Pl)EctA (i) with only N-γ-acetyl-L-2,4-diaminobutyrate (N-γ-ADABA), and
not its isomer N-α-acetyl-L-2,4-diaminobutyrate (N-α-ADABA), as product. (ii) In the negative control
sample (reaction mix, without enzyme) no ADABA can be detected. As references, chemical
synthesized (iii) N-α--ADABA and (iv) N-γ-ADABA were used. The velocity of (Pl)EctA at increasing
concentrations of the substrates B, DAB, and C, acetyl-CoA, was determined. Error bars represent the
standard deviation calculated from two biological and two technical replicas each.
28

Figure 3. Overall-fold of the (Pl)EctA monomeric subunit. A, Cartoon representation of the (Pl)EctA
monomeric subunit including the nomenclature of the secondary structure elements and the tertiary
structure. B, Schematic illustration of the secondary structure of (Pl)EctA.
29

Figure 4. Dimer assembly of the (Pl)EctA enzyme. A, Surface presentation of the dimer assembly of
the (Pl)EctA protein displaying the binding site for the ligands CoA (red) and DAB (blue). B, Illustration
of the dimeric interface, highlighting the protrusion of the side-chain of Tyr38 from monomer B into
the DAB-binding site of monomer A (and vice versa). This graphical representation of the dimer
assembly was rendered by using the (Pl)EctA:CoA:DAB tertiary crystal structure (PDB entry 6SLL) as
the template.
30