Studies on structure-function relationships of acetolactate decarboxylase from: Enterobacter cloacae

Article abstract of DOI:10.1039/c8ra07379a

Acetoin is an important bio-based platform chemical with wide applications. Among all bacterial strains, Enterobacter cloacae is a well-known acetoin producer via α-acetolactate decarboxylase (ALDC), which converts α-acetolactate into acetoin and is identified as the key enzyme in the biosynthetic pathway of acetoin. In this work, the enzyme properties of Enterobacter cloacae ALDC (E.c.-ALDC) were characterized, revealing a K_m value of 12.19 mM and a k_cat value of 0.96 s^-1. Meanwhile, the optimum pH of E.c.-ALDC was 6.5, and the activity of E.c.-ALDC was activated by Mn²⁺, Ba²⁺, Mg²⁺, Zn²⁺ and Ca²⁺, while Cu²⁺ and Fe²⁺ significantly inhibited ALDC activity. More importantly, we solved and reported the first crystal structure of E.c.-ALDC at 2.4 ? resolution. The active centre consists of a zinc ion coordinated by highly conserved histidines (199, 201 and 212) and glutamates (70 and 259). However, the conserved Arg150 in E.c.-ALDC orients away from the zinc ion in the active centre of the molecule, losing contact with the zinc ion. Molecular docking of the two enantiomers of α-acetolactate, (R)-acetolactate and (S)-acetolactate allows us to further investigate the interaction networks of E.c.-ALDC with the unique conformation of Arg150. In the models, no direct contacts are observed between Arg150 and the substrates, which is unlikely to maintain the stabilization function of Arg150 in the catalytic reaction. The structure of E.c.-ALDC provides valuable information about its function, allowing a deeper understanding of the catalytic mechanism of ALDCs.

Full text of DOI:10.1039/c8ra07379a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Studies on structure-function relationships of
acetolactate decarboxylase from Enterobacter
cloacae
Cite this: RSC Adv., 2018, 8, 39066
a
*
Fangling Ji,
Jingyun Wang,
†
Yanbin Feng,†b Mingyang Li,a Yongliang Yang,a Tianqi Wang,a
a
b
Yongming Baoac and Song Xue
*
Acetoin is an important bio-based platform chemical with wide applications. Among all bacterial strains,
Enterobacter cloacae is a well-known acetoin producer via a-acetolactate decarboxylase (ALDC), which
converts a-acetolactate into acetoin and is identified as the key enzyme in the biosynthetic pathway of
acetoin. In this work, the enzyme properties of Enterobacter cloacae ALDC (E.c.-ALDC) were
characterized, revealing a Km value of 12.19 mM and a kcat value of 0.96 sꢀ1. Meanwhile, the optimum pH
of E.c.-ALDC was 6.5, and the activity of E.c.-ALDC was activated by Mn2+, Ba2+, Mg2+, Zn2+ and Ca2+
,
while Cu2+ and Fe2+ significantly inhibited ALDC activity. More importantly, we solved and reported the
first crystal structure of E.c.-ALDC at 2.4 A˚ resolution. The active centre consists of a zinc ion
coordinated by highly conserved histidines (199, 201 and 212) and glutamates (70 and 259). However,
the conserved Arg150 in E.c.-ALDC orients away from the zinc ion in the active centre of the molecule,
losing contact with the zinc ion. Molecular docking of the two enantiomers of a-acetolactate, (R)-
acetolactate and (S)-acetolactate allows us to further investigate the interaction networks of E.c.-ALDC
with the unique conformation of Arg150. In the models, no direct contacts are observed between
Arg150 and the substrates, which is unlikely to maintain the stabilization function of Arg150 in the
catalytic reaction. The structure of E.c.-ALDC provides valuable information about its function, allowing
a deeper understanding of the catalytic mechanism of ALDCs.
Received 4th September 2018
Accepted 15th November 2018
DOI: 10.1039/c8ra07379a
rsc.li/rsc-advances
separation of high purity acetoin from the mixtures is very
expensive.
Introduction
To date, many bacterial strains, such as Bacillus subtilis,1
In microbes, acetoin (3-hydroxy-2-butanone, AC) is the in-
between product of the 2,3-butanediol (BD) fermentation
pathway. With the growing demand in recent years, the
production method of AC has been widely addressed. At
present, commercially available AC is mainly chemically
synthesized from fossil materials, such as 2,3-butanediol (2,3-
BD), butanone and diacetyl. However, due to environmental
problems in chemical processes, large-scale production of AC is
still limited. Based on microbial fermentation and enzymatic
catalysis, biotechnological production provides an alternative
environmentally friendly method, but the nal product is
usually a mixture. The mixture contains by-products, such as
2,3-BD, acetic acid and lactic acid, resulting in the difficult
separation of AC in microbial fermentation. Usually, the
Klebsiella pneumoniae,2 Enterobacter aerogenes3 and Enterobacter
cloacae,4 have been discovered and engineered to produce AC.
Among these microorganisms, the most promising microor-
ganism for biological production of AC is Enterobacter cloacae.
Enterobacter cloacae subsp. dissolvens strain SDM grows rapidly
in a simple medium and metabolizes the major lignocellulose-
derived sugars glucose and xylose into AC.4 However, AC exists
in two stereoisomeric forms: (R)-AC and (S)-AC. The efficient use
of AC, especially in the asymmetric synthesis of valuable chiral
chemicals, requires optically active AC. However, owing to a lack
of knowledge about stereoisomer formation mechanisms, the
production of optically active AC using these strains has been
difficult to achieve. Industrial chiral AC production by using
these promising microorganisms has not been reported to date.
Three enzymes involved in AC biosynthesis from pyruvate
through a-acetolactate (AL) to AC include a-acetolactate syn-
thase (ALS, encoded by budB), a-acetolactate decarboxylase
(ALDC, encoded by budA), and acetoin reductases (ARs, also
called DRs or BDHs, encoded by budC and gdh) (Scheme 1).
ALDC (EC 4.1.1.5) catalyses a-acetolactate to form AC and
carbon dioxide via decarboxylation.5 In some cases, the
aSchool of Life Science and Biotechnology, Dalian University of Technology, Dalian,
Liaoning, 116024, P. R. China. E-mail: fanglingji@dlut.edu.cn
bMarine Bioengineering Group, Dalian Institute of Chemical Physics, Chinese Academy
of Sciences, Dalian, Liaoning, 116023, P. R. China. E-mail: xuesong@dicp.ac.cn
cSchool of Food and Environment Science and Engineering, Dalian University of
Technology, Panjin, Liaoning, 12422, P. R. China
† These two authors contributing equally to the paper.
39066 | RSC Adv., 2018, 8, 39066–39073
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
from the active site, forming hydrogen bonds with residues at
the C-terminus. Additionally, Arg150 was proven to maintain
the same conformation and showed no contacts with the
substrates though molecular docking complexes of E.c.-ALDC
with (S)-AL and (R)-AL. The fully characterized activities and
structure of the enzyme provide insights into ALDCs for the
bioproduction of acetoin.
Scheme 1 Scheme of the acetolactate pathway starting with pyruvate.
In the figure: ALS, a-acetolactate synthase; ALDC, a-acetolactate
decarboxylase; BDH, 2,3-butanediol dehydrogenase; AR, acetoin
reductase; DAR, diacetyl reductase and NOD, nonoxidize non-enzy-
matic oxidative decarboxylation.
Results and discussion
Enzymatic and kinetic properties of E.c.-ALDC
The activity of ALDC was previously studied using the Voges–
Proskauer (VP) assay.29 However, it was challenging to obtain
reproducible results using this assay since the substrate and the
colour regent are not stable during the reaction. To avoid this
problem, we carried out an enzymatic assay on B.s.-ALDC using
CD as our research technique.13 The CD spectra of (S)-AL and
(R)-AC are reported30–32 and are sufficiently different to allow for
the monitoring of the enzyme activity assay. At the beginning,
no CD signal was observed due to the racemic substrate,
whereas the spectra for (R)-AL and (R)-AC appeared in addition
to the enzyme. The signal for (R)-AL was observed because the
enzymatic turnover of (S)-AL produces an excess of (R)-AL
(Fig. 1A). Two wavelengths at 278 nm and 315 nm were identi-
ed to monitor the assay reaction. As presented in Marlow's
work, at 278 nm, the formation of (R)-AC can be monitored. At
this point, the CD signal for (S)-AL crosses the axis and has no
signal. Though the slope is negative because of the negative
peak, but it is proportional to the formation rate of (R)-AC. At
decarboxylation of a-acetolactate by ALDC is assumed to be
a rate-limiting step in generating 2,3-BD.6,7 Immobilized ALDC
has been applied in the traditional beer brewing process.8
During fermentation, ALDC signicantly increases the produc-
tion rate and decreases the adverse avour caused by diacetyl.9
Apart from the catabolic degradation of a-acetolactate to ace-
toin, ALDC also participates in the biosynthesis of branched-
chain amino acids.10–12
Up to now, the functionally characterized and structurally
solved ALDCs are those from Bacillus subtilis (B.s.-ALDC),13
Bacillus brevis (B.b.-ALDC),14 Brevibacillus brevis9 and Lactococcus
lactis subsp. lactis.11 X-ray crystal structures of ALDCs from
Bacillus subtilis13 and Bacillus brevis15 show that both of the
ALDCs feature a two-domain a/b tertiary structure, in which the
N-terminal domain is composed of a seven-stranded mixed b-
sheet. In the asymmetric unit cell, the dimer assembly is
composed of the seven-stranded mixed b-sheet, which extends
into the equivalent b-sheet of the two-fold symmetry-related
molecule, forming a fourteen-stranded b-sheet. The structures
of a series of designed transition state analogues in complex
with B.b.-ALDC suggest a catalytic mechanism. ALDC catalyses
not only the decarboxylation of the favoured (S)-AL but also the
isomerization via carboxyl migration and subsequent decar-
boxylation of the less-favoured (R)-substrate.15 In addition, two
groups16 simulated the catalytic processes using the hybrid
quantum mechanical/molecular mechanical (QM/MM) calcu-
lations. Even though the amino sequence identities between
B.b.-ALDC and B.s.-ALDC are very low (only 30%), the active
centre is well-conserved. By docking the two enantiomers of AL
into the crystal structure of B.s.-ALDC, Ji et al. simulated the
binding interaction of the substrates and investigated the
substrate preference. A more stable binding of (S)-AL versus (R)-
AL is revealed by the lower binding free energy.13
Due to less structural knowledge of ALDCs, the determina-
tion of the structural characteristics of the E.c.-ALDC is essential
to obtain new insights into its catalytic reaction. We thus
cloned, expressed and characterized the ALDC from Enter-
obacter cloacae. Here, we report the crystal structure of E.c.-
Fig.
1 Enzymatic properties of E.c.-ALDC measured by circular
dichroism (CD). (A) E.c.-ALDC catalysed (ꢁ)-AL monitored by contin-
uous wavelength scan CD from 190 nm to 340 nm. The entire spectra
are shown starting from (ꢁ)-AL (blue) to (R)-AC (purple). Different
colour represents different time points. (B) AC signal at 278 (left) and
315 nm (right). At 278 nm, there is no signal for (S)-AL, and the changed
CD signal is due to the formation of (R)-AC. At 315 nm, there is no
signal for (R)-AC, and the changed CD signal is due to the disap-
pearance of (S)-AL.
˚
ALDC at a resolution of 2.4 A, which provides an original view of
the structural features of E.c.-ALDC and investigate the
structure-function relationship of E.c.-ALDC. In the active site,
the conserved His199, His201 and His212 residues preserve the
overall structure of the ALDC, except Arg150, which orients away
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 39066–39073 | 39067

View Article Online
RSC Advances
Paper
315 nm the CD signal for (R)-AC is zero and only the tail of
a positive (S)-AL peak is observed. This is the disappearance of
a positive peak so the rate observed reects the loss of (S)-AL
over time. At 278 nm, the formation of (R)-AL can be observed
initially at a faster rate, followed by a slower rate (Fig. 1B, le).
At 315 nm, the (ꢁ)-AL signal initially started at zero, and the
rapid formation of a negative peak formed due to an excess of
(R)-AL from the enzymatic turnover of (S)-AL. Subsequently, the
peak begins to disappear at a slower rate as the enzyme turnover
for the remaining (R)-AL slows (Fig. 1B, right). As the signal for
(R)-AC was stronger than that for (ꢁ)-AL, the activity was
calculated using the signals at 278 nm, and the specic activity
of E.c.-ALDC (ꢁ)-AL was 1024.37 U mgꢀ1. The rate was converted
from units of mdeg sꢀ1 into units of mM sꢀ1 by using molar
ellipticity and then into specic activity with units of mmol
mgꢀ1 minꢀ1
.
Kinetic studies were conducted on E.c.-ALDC over a concen-
tration range of (ꢁ)-AL. Kinetic data were calculated based on
curve tting to the Lineweaver–Burk double reciprocal curve for
three replicates. The calculated Michaelis–Menten constant
(Km) and turnover number (kcat) values of E.c.-ALDC were
12.19 mM and 0.96 sꢀ1, respectively. In our previous work
studying B.s.-ALDC, the Km and kcat values of B.s.-ALDC were
21 mM and 2.21 sꢀ1
. However, in the kinetic analysis of B.s.-
13
ALDC, Marlow and his coworker15 simultaneously measured
B.s.-ALDC kinetic parameters by the NADH method with (ꢁ)-AL
and the CD method with (S)-AL, yielding Km ¼ 0.25 ꢁ 0.08 mM
and kcat ¼ 5.99 ꢁ 0.95 sꢀ1 for the former and Km ¼ 4.31 ꢁ
1.14 mM and kcat ¼ 29.59 ꢁ 2.96 sꢀ1 for the latter. ALDC activity
has been detected only among certain bacterial species, such as
Bacillus sp. and Lactobacillus sp.,33 and the ALDCs from
different bacteria displayed different physical and chemical
properties. In the thermophilic cell-free cascade enzymatic
reaction for acetoin synthesis from pyruvate, Jia et al. used
creatine and a-naphthol with the Vmax and Km values for pyru-
vate by B.s.-ALDC were 2.34 ꢁ 0.03 mmol minꢀ1 mgꢀ1 and 60.51
ꢁ 2.67 mM, respectively, under the optimum conditions.34
Fig. 2 Optimization of the catalytic conditions of E.c.-ALDC. (A)
Effects of pH on the enzyme activity of E.c.-ALDC. Error bars indicate
standard deviations of three independent experiments. (B) Represen-
tation of the electrostatic potential at the surface of E.c.-ALDC in two
orientations. The protein is shown as solvent-accessible surface col-
oured by electrostatic potential at ꢁ 5 kT eꢀ1. Color-codes depend on
the electrostatic potential (red: negative charge; blue, positive charge;
and white: neutral charge). (C) Effects of different metal ions on the
enzyme activity of E.c.-ALDC. Error bars indicate standard deviations
from three independent experiments.
Optimization of the decarboxylation conditions
calculated theoretical isoelectric point (pI) of E.c.-ALDC was 5.4.
The E.c.-ALDC revealed two differently charged faces. One face
was mostly neutral with two negatively charged areas, while the
second face was highly negatively charged.
To test the impact of pH changing the enzyme activity of E.c.-
ALDC, the activity assays were carried out with pH levels ranging
from 4.5 to 8.5, and the results are presented in Fig. 2A. The
highest activity of E.c.-ALDC enzyme was observed at pH 6.5,
and the activity was 963 ꢁ 28 U mgꢀ1. At pH values ranging from
4.5 to 6.0, the activity of E.c.-ALDC dropped dramatically. As
indicated by the results, E.c.-ALDC activity is more effectively
inhibited at an acidic pH than at an alkaline pH. These results
suggest that the secondary interaction (ionic and polar inter-
actions, hydrogen bonding) within the enzyme was stable,
resulting in improved ALDC tolerance to alkaline conditions.35
The ALDC from Bacillus subtilis exhibited the maximum activity
at pH 5.0.13 The ALDC from Lactobacillus casei DSM 2547
showed a relatively high activity at pH 4.5 to 5.0.36 The ALDC
from Lactococcus lactis had an optimum pH of pH 6.0.37 More-
over, the electrostatic surface properties of E.c.-ALDC were
investigated (Fig. 2B) with an environmental pH of 7.0. The
As is reported in the crystal structures of ALDCs, Zn2+ plays
a very important role in stabilizing the substrates and is
involved in electron transfer. The effects of various metal ions
on E.c.-ALDC were investigated. EDTA was used to chelate the
metal ions of E.c.-ALDC, which were then measured by an
inductively coupled plasma (ICP) technique. As indicated in
Fig. 2C, Mn2+, Ba2+, Mg2+, Zn2+ and Ca2+ greatly improved the
activity of E.c.-ALDC, while Fe2+ inhibited the activity of E.c.-
ALDC. Cu2+ signicantly decreased the activity of E.c.-ALDC. In
fact, the function of many decarboxylases requires a divalent
ion as a cofactor. ALDC from Lactococcus lactis DX was activated
by Fe2+, Zn2+, Mg2+, Ba2+ and Ca2+ and was signicantly
inhibited by Cu2+
. Some of the metal ions do not function as
38
catalytic centres. For example, the Zn2+ ion in the biotin-
39068 | RSC Adv., 2018, 8, 39066–39073
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
dependent decarboxylases helped to properly orient the decarboxylase,5 copper in quercetin 2,3-dioxygenase,43 and zinc
substrate, while the Mg2+ ion in the ThDP-dependent decar- in carbonic anhydrase.44 In the transition state, analogue
boxylases assisted in binding the thiamine cofactor.39,40 For structures of B.b.-ALDC with (2S,3S)-2,3-dihydroxy-2-
NAD(P)+-dependent decarboxylases, such as malic enzymes, methylbutanoic acids (PDB ID: 4BT4) and (2S,3R)-2,3-
a divalent metal ion (Mn2+ or Mg2+) presented signicant dihydroxy-2-methylbutanoic acids (PDB ID: 4BT5), the Glu65,
inuences on both catalysis and structural stability.41
Arg145, and Glu253 residues contact the analogues through
hydrogen bonds, and the inhibitors adopt essentially identical
conformations. Meanwhile, Glu65 and Arg145 also form stabi-
lizing hydrogen bonds with the analogues.15
Overall structure of E.c.-ALDC
The crystal structure of E.c.-ALDC comprises two domains of a/
b tertiary structure, in which each domain contains a seven-
stranded mixed b-sheet. This is essentially the same as the
already described crystal structures of B.s.-ALDC13 and B.b.-
ALDC.42 These two b-sheets form a nearly parallel intra-
molecular surface (back-to-back) (Fig. 3A). In-between this
surface, the shortest distance of all Ca atoms of the backbone
Comparisons of ALDC structures
We compared the structures of E.c.-ALDC, B.b.-ALDC (PDB ID:
4BT2) and B.s.-ALDC (PDB ID: 5NXE). Despite the low sequence
identities of E.c.-ALDC with B.s.-ALDC and B.b.-ALDC, the
superimposition of the overall structure of E.c.-ALDC onto that
of B.b.-ALDC (Fig. 4B, upper le) and B.s.-ALDC (Fig. 4B, upper
˚
chain is 6.2 A between Ile72 and His201. The N-terminal
domain consists of a seven-stranded mixed b-sheet, whereas
the C-terminal domain consists of a ve-stranded b-sheet, two
of which elongate with a 180 degree turn generating two b-sheet
strands. Two ALDC molecules are present in the asymmetric
unit, and they exhibit an average pairwise backbone atom root
˚
right) yields an average pairwise RMSD value of 0.712 A and
˚
0.752 A, respectively, for the backbone atoms. The evolutionary
tree of these three ALDCs is shown in Fig. 4A. The low RMSD
˚
mean square deviation (RMSD) value of 0.26 A, exhibiting
highly structural consistency. The seven-stranded mixed b-sheet
on the N-terminal domain of E.c.-ALDC extends into the
equivalent b-sheet of the two-fold symmetry-related molecule,
resulting in a fourteen-stranded b-sheet that spans the physio-
logically relevant dimeric assembly, with the same structures as
B.b.-ALDC42 and B.s.-ALDC,15 which might shed light on the
dimer formation (Fig. 3A).
In the structure of E.c.-ALDC, the electron density for the
active centre residues is well resolved (Fig. 3B). A Zn2+ ion
coordinated by His199, His201 and His212 together with
Glu259 from the C-terminal tail, and these residues are highly
conserved in ALDCs (Fig. 2B). In addition, the other two highly
conserved Glu70 and Arg150 are also presented in this struc-
ture. In the catalytic domain of B.s.-ALDC, molecular docking
results exhibit a similar conservative structure, a Zn2+ ion
coordinated by His191, His193 and His201. Glu251, Glu62 and
Arg142 interact with the substrate through hydrogen bonds.
Three histidine-coordination of a metal ion is widely found in
the structures of enzymes, for instance, manganese in oxalate
Fig. 4 Crystal structure comparisons within E.c.-ALDC, B.s.-ALDC and
B.b.-ALDC. (A) The crystal structure of E.c.-ALDC (grey) is super-
imposed onto B.b.-ALDC (pink, PDB ID: 4BT7) and B.s.-ALDC (yellow,
PDB ID: 5XNE). (B) Active site superimposed between E.c.-ALDC and
B.b.-ALDC (pink, PDB ID: 4BT7) and between E.c.-ALDC and B.s.-
ALDC (yellow, PDB ID: 5XNE), including Thr63, His199, His201, His212,
Glu70, Glu255, and Arg150. (B.b.-ALDC: Thr58, His194, His196, His207,
Fig. 3 3D structure of E.c.-ALDC. (A) The overall fold of E.c.-ALDC is Glu65, Glu254, and Arg145), (B.s.-ALDC: Thr55, His191, His193, His204,
shown as a ribbon diagram. (B) The electron densities of His199, Glu62, Glu251, and Arg142). (C) Intramolecular interactions of Arg150
His201 and His212; Glu70 and Glu255; and Arg150, which are in (grey, E.c.-ALDC; Arg145, pink, B.b.-ALDC; Arg142, yellow, B.s.-ALDC)
contact with a zinc ion within a 5 A˚ distance.
with Val258, Glu259 and Asn260.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 39066–39073 | 39069

View Article Online
RSC Advances
Paper
value indicates that the overall structure of the E.c.-ALDC is
similar to the structures of B.b.-ALDC and B.s.-ALDC. We also
superimposed the highly conserved amino acid residues in the
active site (His199, His201 His212, Glu70, Glu259, Arg150 and
Thr63) onto those of B.b.-ALDC (Fig. 4B, lower le) and B.s.-
ALDC (Fig. 4B, lower right). Apart from Arg150, the coordinates
of the side chains of the remaining residues overlay well.
The orientation of the side chain of Arg150 in the structure of
E.c.-ALDC differs from the arginine in B.s.-ALDC and in B.b.-
ALDC. In E.c.-ALDC, the side chain of Arg150 orients away from
the active site centre with an angle of 50 degrees. In addition to
the highly conserved Thr58 and Glu65 in B.b.-ALDC, arginine at
the corresponding site is also highly conserved in the vicinity of
the metal. Likewise, in E.c.-ALDC, the distance between the zinc
˚
ion and the NH2 group of the side chain of Arg150 is 8.6 A;
however, in B.s.-ALDC and B.b.-ALDC, this distance is extremely
˚
˚
smaller, 4.0 A and 3.9 A, respectively. In fact, both of the NH2
groups in the side chain of the arginine residue (Arg142 in B.s.-
ALDC and Arg145 in B.b.-ALDC) forms hydrogen bonds with the
oxygen atom of the glutamate residue (Glu250 in B.s.-ALDC and
Glu253 in B.b.-ALDC). In E.c.-ALDC, the NH2 group of Arg150
forms a hydrogen bond with the oxygen atom of the backbone of
˚
Val258 (3.2 A). The NH2 group of Arg150 is in close proximity to
˚
the ND2 group of Asn260 with a distance of 2.9 A (Fig. 4C). It is
possible that the unique conformation of Arg150 causes a loss
of function in stabilizing the substrate.
The glutamate at the active centre plays a signicant role
during the decarboxylation reaction. The highly conserved
Glu259 in E.c.-ALDC conducted a similar structure conforma-
tion as the corresponding glutamate structure in B.s.-ALDC and
B.b.-ALDC. The distance between the carbonyl group of the side
Fig.
5
Energy optimized active site structures of E.c.-ALDC in
complex with the (S)-AL (A, orange) and (R)-AL (B, yellow). Selected key
distances are shown in angstroms. Hydrogen bonds are shown in black
dashed lines, and coordination bonds are shown in red dashed lines.
˚
chain and the zinc ion of Glu259 is 3.6 A, while in the structures
˚
˚
of B.s.-ALDC and B.b.-ALDC, the distances are 3.5 A and 4.0 A,
respectively. In the crystal structure of B.b.-ALDC in complex
with transient intermediate analogues, glutamate forms
a hydrogen bond with the analogues.15 Meanwhile, in the
docking models of B.b.-ALDC with (S)-AL, the carboxyl group of
Glu253 forms a hydrogen bond with Arg145 in the active site.9 In
the rearrangement process, the hydrogen bond between Glu253
and the carboxyl of (R)-AL is critical when the carboxyl rear-
ranges to the adjacent carbonyl carbon to form (S)-AL.16
method. The role of the Zn(II), as a Lewis acid, is best under-
stood in terms of the reverse reaction of acetoin carboxylation.
The mode of binding for the (R,R)-dihydroxy-2-methylbutanoic
acids (PDB ID: 4BT5) isomer supports the proposed decarbox-
ylation mechanism for the natural (S)-enantiomer, also
involving transient binding of the departing CO2 to the zinc ion.
In addition, the side chain of Glu70 (Glu65 in B.s.-ALDC and
Glu62 in B.b.-ALDC) has polar interactions with (S)-AL. Like-
wise, Arg150 maintains the interactions with Val258, Glu259
and Asn260 as in the apo structure of E.c.-ALDC and did not
change its unique conformation in the presence of (S)-AL. Thus,
Arg150 is not able to preserve the critical role in stabilizing the
Zn2+ ion, which differs from the corresponding arginines in
B.b.-ALDC and B.s.-ALDC. In the transition state, analogue
structures of B.b.-ALDC in complex with (2S,2S)-2,3-dihydroxy-2-
methylbutanoic acid (PDB ID: 4BT4) and (2S,2R)-2,3-dihydroxy-
2-methylbutanoic acid, Arg145 contacts the analogues through
hydrogen bonds, and the inhibitors adopt essentially identical
conformations. Arg145 is well positioned to potentially assist
delivery of the proton, which creates a new chiral centre in B.b.-
ALDC.15 In the docking model of B.s.-ALDC with (S)/(R)-AL,
a hydrogen bond between the OH of (R)-AL and the side chain of
Arg142 is observed, while no hydrogen bond formed in B.s.-
ALDC with (S)-AL.13 In the transition state of analogue
Molecular docking with substrate
The complex structures of E.c.-ALDC with (S)-AL and (R)-AL were
prepared on the basis of crystal structures of B.b.-ALDC in
complex with two transition state analogues. The energy opti-
mized structures are displayed in Fig. 5. In the complex struc-
ture of (S)-AL and E.c.-ALDC, the Zn2+ ion forms
a pentacoordinated structure with the nearby His199, His201
˚
and His212 at distances of 1.9, 2.0 and 2.2 A, respectively, and
with the oxygen atoms on the carboxyl and hydroxyl groups of
˚
(S)-AL at distances of 1.9 and 2.7 A, respectively; this indicates
a strong coordination interaction of the Zn2+ ion with the three
histidine residues and the substrate (Fig. 5A). These interaction
networks are also presented in the models of B.s.-ALDC with (S)-
AL13 and B.b.-ALDC with (S)-AL9,16 via the molecular docking
39070 | RSC Adv., 2018, 8, 39066–39073
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
structures of B.b.-ALDC in complex with (2S,2S)-dihydroxy-2- 6.0 by the addition of HCl and then diluted with 10 mM Tris–
methylbutanoic acid (PDB ID: 4BT4) and (2S,2R)-dihydroxy-2- HCl buffer (pH 6.0) to the desired concentration. To generate
methylbutanoic acid, Arg145 contacts the analogues through the standard curve of (R)-AC, with the complete reaction of
hydrogen bonds, and the inhibitors adopt essentially identical (ꢁ)-AL with E.c.-ALDC, the produced stock solution was diluted
conformations.
to 0.5, 1.0, 5.0, 10.0, 15.0 and 0.5 mM. The standard curve of (R)-
The binding mode of E.c.-ALDC with (R)-AL slightly differs AC was measured by CD using the following parameters: the
from the binding mode of (S)-AL. As shown in Fig. 5B, (R)-AL scanning range 200–340 nm, data pitch 1 nm, response 0.5 s,
adopts a perpendicular conformation to that of (S)-AL in E.c.- bandwidth 2 nm and scanning speed 200 nm minꢀ1. Using
ALDC with its polar groups facing Glu259. The hydroxyl and a cuvette with an optical path length of 1 mm, an equal volume
carboxyl groups of (R)-AL are further from Glu259, forming of substrate and E.c.-ALDC were mixed to start the reaction. The
hydrogen bonds with the OE1 and OE2 groups of Glu259 at subsequent decarboxylation of (ꢁ)-AL into (R)-AC was moni-
a distance of 2.7 and 4.2 A, respectively. Thus, the Zn2+ ion only tored, and the CD signal at 278 nm was recorded. The amount
˚
forms a tetracoordinated structure with His199, His201, His212 of AC produced by ALDC was measured against the (R)-AC
and the substrate, with binding distances of 1.9, 2.0, 2.2 and 2.0 standard curve, resulting in the activity of E.c.-ALDC. One unit
˚
A, respectively. These hydrogen bonds stabilize the substrate in of E.c.-ALDC activity corresponds to the amount of E.c.-ALDC
ꢂ
the active site of the enzyme. Arg150 in this model exhibits no required for the formation of 1 mmol AC per minute at 30 C.
conformational changes without interacting with (R)-AL. Again,
The kinetic parameters (Km and kcat values) of E.c.-ALDC were
Arg150 is unlikely to stabilize the substrate or deliver the proton calculated by measuring enzyme activity using various initial
in the catalytic function of E.c.-ALDC. To our knowledge, (R)-AL concentrations (53.4, 26.7, 17.8, 13.3, 10.7, 8.9, 7.6, 6.7, 5.9 and
is not the natural substrate of ALDCs, while (S)-AL is. Corre- 5.3 mM) of (ꢁ)-AL at 30 ꢂC and pH 6 and tting to a double
spondingly, in the cocrystallized complexes of B.b.-ALDC with reciprocal curve by a Lineweaver–Burk plot. The process was
the substrate analogues, the promoting carboxylate rearrange- monitored by CD as described above.
ment mechanism of (R)-AL into (S)-AL was observed and
Optimum pH and ion determination. The optimal pH of E.c.-
ALDC was determined in buffers with different pH values
(citrate phosphate 4.5–6.5, Tris 6.0–8.5) at 30 ꢂC. E.c.-ALDC was
preincubated with these buffers at 4 C for an hour, and then
the activity of E.c.-ALDC was determined using the CD above-
mentioned method at a temperature of 30 ꢂC. Moreover, the
proposed.42
ꢂ
Experimental
Materials and methods
Expression and purication of E.c.-ALDC. Protein was electrostatic surface potentials of E.c.-ALDC were calculated by
extracted essentially based on procedures previously reported APBS17 using an ionic strength of 0.15 M. The PDB2PQR server18
for ALDCs.13 The genome of Enterobacter cloacae CICC 10011 was used to prepare the structure for electrostatic calculations.
was used as a template to clone the gene of E.c.-ALDC (bud A) Figures were generated using PYMOL 1.5.0.3.19 In addition, the
and inserted into the expression vector pET-21b. E. coli BL21 effect of different metal ions on the activity of E.c.-ALDC was
(DE3)ꢂharbouring pET-21b with the E.c.-ALDC gene were grown analysed with 1 mM Cu2+, Fe2+, Mn2+, Ba2+, Mg2+, Zn2+ and Ca2+
ꢂ
at 37 C and induced for 5 h. E. coli cells were then harvested, by the CD method at 30 C. In advance, E.c.-ALDC was treated
and the pellet was resuspended in 20 mM tris(hydroxymethyl) with 0.5 mg mLꢀ1 EDTA, and the residual metal ions were
aminomethane (Tris) buffer (pH 8.0), containing 1 mM dithio- measured by inductively coupled plasma emission spectrometry
threitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA) (ICP-ES).
and 20% glycerol, followed by lysis by sonication on ice at the
Crystallization. Commercially available crystallization
power of 400 W for 10 min. The supernatant was loaded onto reagents, such as Index (Hampton Research) and Wizard (Agi-
a pre-equilibrated HiTrap DEAE FF anion exchange column (GE lent), were used to set up crystal screening. The initial crystal-
Healthcare) in 20 mM Tris buffer (pH 8.0) containing 1 mM lization trials of E.c.-ALDC were performed using a Mosquito
DTT. The column was then eluted using a linear NaCl gradient crystallization robot (TTP LabTech) to set up sitting drops. The
from 0 to 500 mM over 20 column volumes. Fractions con- drops were composed of 0.1 mL protein solution mixed with an
taining the protein were collected, concentrated and subjected equal volume of reservoir solution equilibrated against 40 mL of
to gel ltration on a Superdex 200 16/60 column (GE Healthcare) the reservoir solution. Small crystals were observed aer 2
ꢂ
in 20 mM Tris buffer (pH 8.0) containing 1 mM DTT. The weeks in the plates at 4 C. The nal crystal for the diffraction
puried protein was conrmed by SDS-PAGE, and the protein collection is from a crystal grown in 0.1 M sodium acetate tri-
concentration was determined using the Bradford protein assay hydrate, pH 4.5, 3.0 M sodium chloride at a protein concen-
method.
Enzyme activity assay. For the activity assay of E.c.-ALDC, the
tration of 30 mg mLꢀ1 at 4 C.
ꢂ
X-ray data collection and processing. Crystals of E.c.-ALDC
measurements were based on procedures previously reported grown in the above conditions were cryoprotected by incubation
for B.s.-ALDCs,13 using circular dichroism (CD, JASCO J-810) to for 30 s in a solution containing glycerol (15%) in 0.1 M sodium
study the activity and kinetics of E.c.-ALDC.15 Freshly prepared acetate trihydrate, pH 4.5, 3.0 M sodium chloride prior to ash-
substrate (ꢁ)-AL was obtained by diluting (ꢁ)-ethyl 2-acetoxy-2- cooling in liquid nitrogen. Datasets were collected at the
methylacetoacetate (Aldrich) with 0.5 M NaOH and stirring for Shanghai Synchrotron Radiation Facility (SSRF) on beamline
20 min at room temperature. The mixture was adjusted to pH BL18U1. Aer the optimization of the procedures, 360
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 39066–39073 | 39071

View Article Online
RSC Advances
Paper
diffraction images were collected using an MX225 charged the basis of the crystal structures of B.b.-ALDC in complex with
coupled device (CCD) detector at 100 K with an oscillation range the two transition state analogues (PDB ID: 4BT4 and 4BT5).
of 1.0ꢂ and an exposure time of 1 s per frame, with a non- Input structures of E.c.-ALDC and (S)/(R)-AL for docking simu-
˚
attenuated beam of 0.98 A X-ray wavelength. All diffraction data lation were stored in the PDBQT le format of AutoDock 4.20.
were processed, integrated, and scaled using the HKL-3000 The ligand input les were prepared according to the standard
soware package.20 The structure was solved by molecular protocols in the AutoDock manual to prepare the protein target
replacement (MR) with Phaser21 using the coordinates of the les. The default grid size was 60 ꢃ 60 ꢃ 60 points, and the
˚
catalytic domain of the same protein as an initial search model spacing was 0.375 A, which corresponds to a cube with an edge
˚
(Protein Data Bank (PDB) ID 5XNE). The model was improved by length of 22.5 A. The centre of the grid box was dened as the
alternating cycles of manual model building and automated centre of the cocrystallized ligand. All the above procedures
renement using Coot22 and rened using REFMAC5 23 and were performed using AutoDock Tools.
PHENIX.24 The stereochemical quality of the nal model was
assessed with MolProbity.25 In total, 96% and 4% of all residues
are located in the favoured and allowed regions of the Ram-
Conclusions
achandran plot, respectively, and no residues were found in the In this study, we report the enzymatic characterizations and
disallowed region. The atomic coordinates and structure factor original structure of ALDC from Enterobacter cloacae. Despite its
for E.c.-ALDC have been deposited in the Protein Data Bank high affinity for substrates at a Km of 12.19 mM, the enzyme
(accession code 5YHO). All structure gures were prepared presented low catalytic efficiency. The structure of E.c.-ALDC
using PyMOL.26 A summary of the data-collection processing preserved the typical conservative conformation of known
and structure-renement statistics is given in Table 1.
ALDCs. Surprisingly, Arg150 exhibits a tilted orientation away
Docking studies of E.c.-ALDC with (S)/(R)-AL. Similar dock- from the active centre and loses its interaction with the zinc ion.
ing investigation of E.c.-ALDC with the substrates was con- Furthermore, by docking the substrates into the structure of
ducted as the previous procedures in AutoDock 4.20 (http:// E.c.-ALDC, no direct contacts are observed between Arg150 and
www.autodock.scripps.edu/).13 The empirical free energy func- the substrates. Thus, we believe that the unique conformation
tion and the Lamarckian genetic algorithm were employed to of Arg150 might explain the lower enzymatic activity of E.c.-
identify the binding sites of (S)/(R)-AL with E.c.-ALDC.27,28 The ALDC compared to that of the previously characterized ALDCs.
computational models used in this work were constructed on However, further experimental cocrystallization studies are
needed in the future to prove our hypothesis and in silico
calculations. Our E.c.-ALDC structure provides very important
clues to the catalytic mechanism of ALDCs.
Table 1 Data collection and refinement statistics for E.c.-ALDCa
Data collection
Space group
P1211
Conflicts of interest
Cell dimensions
There are no conicts of interest to declare.
˚
a, b, c (A)
45.832, 59.981, 80.818
90.00, 108.17, 90.00
64 798 (6054)
16 057 (1551)
47.27–2.40
0.075
23.47
97.69
4.0
a, b, g (ꢂ)
Acknowledgements
Total reections
Unique reections
˚
Feida Long helped with the docking work. Additionally, we
would like to thank the nancial support from the National
Science Foundation of China with grant number 21506025 and
the Dalian University of Technology Science Foundation with
grant number DUT8LK08.
Resolution (A)
(2.49–2.40)
(0.244)
(8.58)
(88.35)
(3.9)
Rmerge
hI/sIi
Completeness (%)
hRedundancyi
Renement
Resolution (A)
˚
47.27–2.40
16 044
0.211/0.272
(2.46–2.40)
(1538)
(0.223/0.251)
Notes and references
No. reections
Rwork/Rfree
1 B. Zhang, X. L. Li, J. Fu, N. Li, Z. Wang, Y. J. Tang and
T. Chen, PLoS One, 2016, 11, e0159298.
2 J. W. Jang, H. M. Jung, D. G. Kim and M. K. Oh, Korean Chem.
Eng. Res., 2017, 55, 237–241.
No. atoms
Protein
Water
3742
111
3 J. W. Jang, H. M. Jung, D. K. Im, M. Y. Jung and M. K. Oh,
Enzyme Microb. Technol., 2017, 106, 114.
4 L. Zhang, Q. Liu, Y. Ge, L. Li, C. Gao, P. Xu and C. Ma, Green
Chem., 2016, 18, 1560–1570.
B-factors
Protein
Water
40.20
37.00
RMSD
5 R. Anand, P. C. Dorrestein, C. Kinsland, T. P. Begley and
S. E. Ealick, Biochemistry, 2002, 41, 7659–7669.
6 S. Li, X. Gao, N. Xu, L. Liu and J. Chen, Microb. Cell Fact.,
2014, 13, 55.
˚
Bond lengths (A)
0.004
0.97
Bond angles (ꢂ)
a
Values for the highest resolution bin are given in parentheses.
39072 | RSC Adv., 2018, 8, 39066–39073
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
7 M. H. Choi, S. J. Kim, J. W. Kim, H. J. Sim, Y. C. Park and 25 V. B. Chen, W. B. A. Iii, J. J. Headd, D. A. Keedy,
J. H. Seo, Bioresour. Technol., 2016, 51, 170–176.
8 S. Qian, C. Wang, H. Wang, F. Yu, C. Zhang and H. Yu,
Process Biochem., 2015, 50, 1388–1393.
R. M. Immormino, G. J. Kapral, L. W. Murray,
J. S. Richardson and D. C. Richardson, Acta Crystallogr.,
2010, 66, 12–21.
9 C. Zhao, H. Su and Y. Liu, RSC Adv., 2016, 6, 80621–80629.
10 N. N. M. C. Renna, L. R. Winik and S. A. Zahler, J. Bacteriol.,
1993, 175, 3863–3875.
26 Schrodinger, LLC, The PyMOL Molecular Graphics System,
Version 1.8., 2015.
27 M. F. Sanner, J. Mol. Graphics Modell., 1999, 17, 57–61.
11 N. Goupilfeuillerat, M. Cocaignbousquet, J. J. Godon, 28 G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey,
S. D. Ehrlich and P. Renault, J. Bacteriol., 1997, 179, 6285–
6293.
W. E. Hart, R. K. Belew and A. J. Olson, J. Comput. Chem.,
2015, 19, 1639–1662.
12 C. Monnet, M. Nardi, P. Hols, M. Gulea, G. Corrieu and 29 B. Diderichsen, U. Wedsted, L. Hedegaard, B. R. Jensen and
V. Monnet, Lett. Appl. Microbiol., 2010, 36, 399–405. C. Sjøholm, J. Bacteriol., 1990, 172, 4315.
13 F. Ji, M. Li, Y. Feng, S. Wu, T. Wang, Z. Pu, J. Wang, Y. Yang, 30 D. H. G. Crout and S. M. Morrey, J. Chem. Soc., Perkin Trans.
S. Xue and Y. Bao, Appl. Microbiol. Biotechnol., 2018, 102, 1–
13.
14 S. Najmudin, J. T. Andersen, S. A. Patkar, T. V. Borchert,
1, 1983, 2435–2440, DOI: 10.1039/P19830002435.
31 A. F. Drake, G. Siligardi, D. H. G. Crout and D. L. Rathbone, J.
Chem. Soc., Chem. Commun., 1987, 24, 1834–1835.
¨
¨
D. H. G. Crout and V. Fulop, Acta Crystallogr., 2003, 59, 32 M. Vinogradov, A. Kaplun, M. Vyazmensky, S. Engel,
1073–1075.
R. Golbik, K. Tittmann, K. Uhlemann, L. Meshalkina,
¨
15 V. A. Marlow, D. Rea, S. Najmudin, M. Wills and V. Fueloep,
ACS Chem. Biol., 2013, 8, 2339–2344.
16 C.-B. Zhuang and Q.-C. Zheng, RSC Adv., 2016, 6, 91852–
91859.
Z. Barak and G. Hubner, Anal. Biochem., 2005, 342, 126–133.
33 S. E. Godtfredsen, H. Lorck and P. Sigsgaard, Carlsberg Res.
Commun., 1983, 48, 239–247.
34 X. Jia, Y. Liu and Y. Han, Sci. Rep., 2017, 7, 4333.
17 N. A. Baker, D. Sept, S. Joseph, M. J. Holst and 35 S. L. Hosseinipour, M. S. Khiabani, H. Hamishehkar and
J. A. Mccammon, Proc. Natl. Acad. Sci. U. S. A., 2001, 98,
10037–10041.
18 T. J. Dolinsky, J. E. Nielsen, J. A. Mccammon and N. A. Baker,
Nucleic Acids Res., 2004, 32, 665–667.
19 W. L. Delano, The PyMOL User's Manual, DeLano Scientic,
San Carlos, CA, USA, 2002.
R. Salehi, J. Nanopart. Res., 2015, 17, 382.
36 A. M. Rasmussen, R. M. Gibson, S. E. Godtfredsen and
M. Ottesen, Carlsberg Res. Commun., 1985, 50, 73–82.
37 K. Ius, V. M. Serebrennikov, N. A. Zagustina and
A. M. Bezborodov, Prikladnaia Biokhimiia I Mikrobiologiia,
2000, 36, 131.
20 Z.
Otwinowski
and
W.
Minor,
Macromolecular 38 Y. Guo, D. Pan, H. Ding, Z. Wu, Y. Sun and X. Zeng, J. Sci.
Food Agric., 2015, 95, 1655–1661.
Crystallography, Pt A, 1997, vol. 276, pp. 307–326.
21 A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, 39 W. Buckel, Methods Enzymol., 1986, 125, 547–558.
M. D. Winn, L. C. Storoni and R. J. Read, J. Appl. 40 C. Vogel and J. Pleiss, Proteins: Struct., Funct., Bioinf., 2014,
Crystallogr., 2007, 40, 658–674.
82, 2523–2537.
22 P. Emsley, B. Lohkamp, W. G. Scott and K. Cowtan, Acta 41 N. Niedzwiecka, J. Gronczewska and E. F. Skorkowski, Fish
Crystallogr., 2010, 66, 486–501.
Physiol. Biochem., 2017, 43, 351–360.
´
¨
¨
23 G. N. Murshudov, P. Skubak, A. A. Lebedev, N. S. Pannu, 42 V. A. Marlow, D. Rea, S. Najmudin, M. Wills and V. Fulop,
R. A. Steiner, R. A. Nicholls, M. D. Winn, F. Long and
ACS Chem. Biol., 2013, 8, 2339–2344.
A. A. Vagin, Acta Crystallogr., 2011, 67, 355–367.
43 F. Fusetti, K. H. Schroter, R. A. Steiner, P. I. van Noort,
T. Pijning, H. J. Rozeboom, K. H. Kalk, M. R. Egmond and
B. W. Dijkstra, Structure, 2002, 10, 259–268.
´
24 P. D. Adams, P. V. Afonine, G. Bunkoczi, V. B. Chen,
I. W. Davis, N. Echols, J. J. Headd, L. W. Hung, G. J. Kapral
and R. W. Grosse-Kunstleve, Acta Crystallogr., Sect. D: Biol. 44 Z. Fisher, J. A. H. Prada, C. Tu, D. Duda, C. Yoshioka,
Crystallogr., 2010, 66, 213–221.
H. Q. An, L. Govindasamy, D. N. Silverman and
R. McKenna, Biochemistry, 2005, 44, 1097–1105.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 39066–39073 | 39073