R-hydroxynitrile lyase from the cyanogenic millipede, Chamberlinius hualienensis—A new entry to the carrier protein family Lipocalines

Article abstract of DOI:10.1111/febs.15490

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into cyanide and the corresponding aldehyde or ketone. Moreover, they catalyze the synthesis of cyanohydrin in the reverse reaction, utilized in industry for preparation of enantiomeric pure pharmaceutical ingredients and fine chemicals. We discovered a new HNL from the cyanogenic millipede, Chamberlinius hualienensis. The enzyme displays several features including a new primary structure, high stability, and the highest specific activity in (R)-mandelonitrile ((R)-MAN) synthesis (7420 U·mg^?1) among the reported HNLs. In this study, we elucidated the crystal structure and reaction mechanism of natural ChuaHNL in ligand-free form and its complexes with acetate, cyanide ion, and inhibitors (thiocyanate or iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55?? resolutions, respectively. The structure of ChuaHNL revealed that it belongs to the lipocalin superfamily, despite low amino acid sequence identity. The docking model of (R)-MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen bonds with R38 and K117, and the nitrile group forms hydrogen bonds with R38 and Y103. The mutational analysis showed the importance of these residues in the enzymatic reaction. From these results, we propose that K117 acts as a base to abstract a proton from the hydroxyl group of cyanohydrins and R38 acts as an acid to donate a proton to the cyanide ion during the cleavage reaction of cyanohydrins. The reverse mechanism would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida). Databases: Structural data are available in PDB database under the accession numbers 6JHC, 6KFA, 6KFB, 6KFC, and 6KFD.

Full text of DOI:10.1111/febs.15490

R-hydroxynitrile lyase from the cyanogenic millipede,
Chamberlinius hualienensis—A new entry to the carrier
protein family Lipocalines
Fumihiro Motojima1,2 , Atsushi Izumi1,2, Aem Nuylert1 , Zhenyu Zhai1,2,
*
, Mohammad
Dadashipour1,2,†
, Sayaka Shichida1,2, Takuya Yamaguchi1,2,‡ , Shogo Nakano1,2,–
and
Yasuhisa Asano1,2
1
2
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama, Japan
Asano Active Enzyme Molecule Project, ERATO, JST, Imizu, Toyama, 939-0398, Japan
Keywords
Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into
cyanide and the corresponding aldehyde or ketone. Moreover, they cat-
alyze the synthesis of cyanohydrin in the reverse reaction, utilized in indus-
try for preparation of enantiomeric pure pharmaceutical ingredients and
fine chemicals. We discovered a new HNL from the cyanogenic millipede,
Chamberlinius hualienensis. The enzyme displays several features including
a new primary structure, high stability, and the highest specific activity in
catalytic mechanism; Chamberlinius
hualienensis; crystal structure; hydroxynitrile
lyase; lipocalin family; millipede; site-
directed mutagenesis
Correspondence
Y. Asano, Biotechnology Research Center
and Department of Biotechnology, Toyama
Prefectural University, 5180 Kurokawa,
Imizu, Toyama 939-0398 JAPAN
Tel. +81 766 56 7500
(R)-mandelonitrile ((R)-MAN) synthesis (7420 UÁmgÀ1
) among the
reported HNLs. In this study, we elucidated the crystal structure and reac-
tion mechanism of natural ChuaHNL in ligand-free form and its com-
plexes with acetate, cyanide ion, and inhibitors (thiocyanate or
E-mail: asano@pu-toyama.ac.jp
ꢀ
iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55 A resolutions, respectively. The
structure of ChuaHNL revealed that it belongs to the lipocalin superfam-
ily, despite low amino acid sequence identity. The docking model of (R)-
MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen
bonds with R38 and K117, and the nitrile group forms hydrogen bonds
with R38 and Y103. The mutational analysis showed the importance of
these residues in the enzymatic reaction. From these results, we propose
that K117 acts as a base to abstract a proton from the hydroxyl group of
cyanohydrins and R38 acts as an acid to donate a proton to the cyanide
ion during the cleavage reaction of cyanohydrins. The reverse mechanism
would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida)
Present address
*Center for Experimental Medicine, The
First Affiliated Hospital of Nanchang
University, Nanchang,Jiangxi, 330006, China
†Faculty of Medicine, Health and Life
Sciences, School of Biological Sciences,
Queen’s University Belfast, 19 Chlorine
Gardens, Malone Road, Belfast, NI, BT9
5DL, UK
‡Faculty of Life and Environmental
Sciences, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki, 305-8572,
Japan
Databases
–Graduate Division of Nutritional and
Environmental Sciences, University of
Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka,
422-8526, Japan
Structural data are available in PDB database under the accession numbers 6JHC, 6KFA,
6KFB, 6KFC, and 6KFD.
Fumihiro Motojima and Atsushi Izumi
contributed equally to this work
Enzyme: EC4.1.2.10.
Abbreviations
HNL, hydroxynitrile lyase; MAN, mandelonitrile; NAG, N-acetylglucosamine; PDI, protein disulfide isomerase; SAD, single anomalous
dispersion.
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Federation of European Biochemical Societies
1
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3D structure of hydroxynitrile lyase from a millipede
F Motojima et al.
(Received 15 April 2020, revised 30 June
2020, accepted 8 July 2020)
doi:10.1111/febs.15490
Introduction
belonging to seven different superfamilies: glucose-
methanol-choline (GMC) oxidoreductase [14], a/b-hy-
drolases [15-17], serine carboxypeptidase [18], cupin
[3], Zn2+-containing alcohol dehydrogenase [19], Bet
v1 [9], and dimeric a + b barrel folds [20]. The struc-
ture of the well-studied HNL from Prunus dulcis is
classified into GMC oxidoreductase fold [14]. R-
PaHNL contains flavin adenine dinucleotide (FAD),
which was shown to be not directly involved in the
enzyme catalysis [21], but having a stabilizing role for
the structure of enzyme. The structure of HNLs from
Manihot esculenta (S-MeHNL) and Hevea brasiliensis
(S-HbHNL) bears similarity to those belonging to a/
b-hydrolase family. Although the a/b-hydrolase type
esterases form an oxyanion hole during the catalysis,
a/b-hydrolase type HNLs do not have such structures
[22]. The HNL from Sorghum bicolor (S-SbHNL)
belongs to the serine carboxypeptidase fold with a
conserved peptidase motif, although it does not exhi-
bit carboxypeptidase activity for potential substrates
[17]. The structure of HNL from Granulicella tundri-
cola (GtHNL), reported as the first crystallized bacte-
rial HNL, belongs to the cupin fold bearing a small
barrel structure as a metal-dependent enzyme [3]. The
structure of the newly found HNL from fern,
DtHNL, is reported to be a Bet v1-like fold, which is
composed of antiparallel b-sheet wrapped by a-helices
[9]. We showed that the HNL from Passiflora edulis
(PeHNL) is the first HNL belonging to dimeric a + b
barrel (DABB) superfamily, and it is the shortest
HNL ever known [20]. These HNLs are considered
to have evolved from different ancestral proteins as
expanding example of convergent evolution and using
different catalytic residues for the cleavage and syn-
thesis of cyanohydrins [2]. There are still more HNLs
reported with unknown tertiary structures, such as
the one similar to oxidoreductase containing Zn2+
[19], the one with a new primary structure [23], etc.
In 2015, our group discovered an HNL from the
Hydroxynitrile lyases (HNLs) have been known to
occur mostly in plants [1,2], and recently, those from
bacteria [3,4] and arthropod (cyanogenic millipedes)
have been newly added to the group [5,6]. HNLs of
plant origin have been studied well and their new dis-
tribution, characterization, and structure determination
are reported. We and others discovered HNL activities
by screening natural plants [2,7,8]. They include S-
stereoselective HNL in the leaves of Baliospermum
montanum and R-HNLs from Chaenomles sinensis,
Sorbus aucuparia, Prunus persica, Eriobotrya japonica
and recently from fern Davallia tyermannii [9], etc.
Metal-dependent HNLs were newly discovered and
characterized from bacteria [3,4]. The plant and milli-
pede enzymes catalyze the last step of cyanogenesis in
nature in which a cyanohydrin (alpha-hydroxynitrile)
is cleaved into HCN and the corresponding aldehyde
or ketone. This process is considered to function as
one of their defense mechanism against herbivores and
microbes, respectively [10]. On the other hand, HNLs
are attractive biocatalysts for the synthesis of optically
active cyanohydrins as valuable building blocks for the
synthesis of pharmaceuticals and agrochemicals, since
they catalyze asymmetric syntheses with either of R- or
S-stereoselectivities by an addition of one carbon and
the formed chiral cyanohydrin are versatile compound
modified into varieties of synthetic intermediates by
several chemical procedures [11].
€
Wohler and Liebig studied ‘emulsin’, hydrolyzing
amygdalin to HCN, suggesting an occurrence of pos-
sible b-glucosidase and HNL in Prunus dulcis (syn.,
P. amygdalus: PaHNL) in 1832 [12]. HNLs have been
purified and characterized from several plants and
bacteria, by recent discoveries which have been made
since the interesting aspects of biological cyanogenesis
[13] and the potential of HNL as industrial catalysts
had been recognized by several researchers. From the
structural point of view, the class of the enzymes is
now known to have several tertiary structures, proba-
bly because relatively simple single base mechanisms
would allow the ancestral proteins to evolve to
HNLs. The crystal structures of HNLs in Protein
Data Bank (PDB) exhibit similarity to those
invasive
millipede,
Chamberlinius
hualienensis
(ChuaHNL). ChuaHNL showed the highest specific
activity during (R)-MAN synthesis among the
reported HNLs [5]. Since ChuaHNL has no similarity
to other HNLs or any sequences in the BLAST
2
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F Motojima et al.
3D structure of hydroxynitrile lyase from a millipede
database, its structure and the catalytic mechanism
have not been predicted.
interactions formed among residues in b2-b4 (Fig 1C).
There were two salt bridges between the carboxyl group
of the C-terminal end (Y162) in one molecule and H81 in
another molecule (Fig. 1C). There were three intramolec-
ular disulfide bonds between C3-C108, C35-C152, and
C104-C118 and two intermolecular disulfide bonds
between C80 of one molecule and C158 of another
(Fig 1B and 1C). The root-mean-square deviation
In this study, we successfully determined the crystal
structure of ChuaHNL purified as a dimer form from
the millipedes in ligand-free form, its complexes with
acetate, cyanide ion, and inhibitors (thiocyanate or
ꢀ
iodoacetate) determined at 1.6, 1.5, 2.1, 1.55 A resolu-
tion, respectively. A structure similarity search indicated
that the overall structure of ChuaHNL is similar to lipo-
calin fold proteins even with low-sequence identities.
The docking simulation of (R)-MAN and the kinetic
parameters of the mutational study using recombinant
ChuaHNL expressed in Pichia pastoris suggested that
K117 abstracts a proton from the hydroxyl group and
R38 donates a proton to the released cyanide ion during
the cleavage reaction of cyanohydrins. Therefore,
ChuaHNL follows the acid/base catalysis mechanism of
action during the cleavage and synthesis reactions. This
research reports the first crystal structure of an HNL
other than those from plants and bacteria, from a cya-
nogenic millipede. We present various forms of the crys-
tal structures of the enzyme possessing both new
primary and tertiary structures. 3D structural determi-
nation of this biocatalyst will assist improving its func-
tion for any potential application as well as reveals a
wider range of protein scaffolds with HNL activity in
the nature as a great example of convergent evolution.
ꢀ
(RMSD) among whole structure was 0.13 A at 162 Ca
atoms. Therefore, there was no significant change in the
structure of ChuaHNL upon ligand binding.
Although no homologous sequence with ChuaHNL
has been found in BLAST search [5], the structure
comparison search using DALI server [24] revealed
that the structure of ChuaHNL resembles those of lipo-
calin family proteins. Many typical lipocalins gave Z-
scores higher than 10, indicating significant structural
similarity. The Z-scores is a composite of several evalu-
ations including RMSD of three-dimensional alignment
and numbers of residues matched. A Z-score of more
than 10 is typically given to a match of very strong sim-
ilarities [25]. The secondary structure-based multiple
sequence alignment between ChuaHNL and typical
lipocalins is shown in Fig. 2A. Overall amino acid
sequence identity of ChuaHNL to that of lipocalins
was less than 8%. Lipocalins generally contain three
structurally and sequentially conserved motifs known
as structurally conserved regions (SCRs) 1 to 3 [26].
Even though the amino acid similarity among the
SCRs was very low (Fig. 2A), the secondary structure
of ChuaHNL overlapped well with that of human reti-
nol binding protein 4, RBP4 (Fig. 2B).
Results
Overall structure of ChuaHNL
The crystal structure of the natural ChuaHNL, purified
from the millipedes, was determined by SAD method
using an iodine-soaked ChuaHNL crystal diffracted to
Previous research has indicated that the natural
ChuaHNL is glycosylated [5]. In the thiocyanate-bound
form, N-acetylglucosamine (NAG) moiety with an (a1,
6)-fucose was observed at N123 (Fig. S1A). In other
structures of ChuaHNL, one well-ordered NAG moiety
linking N109 and N123 was observed (Fig. S1B and
S1C). The recombinant ChuaHNL has been also con-
firmed that it was glycosylated at the same glycosylation
sites (at position N109 and N123) in ChuaHNLs pro-
duced in Pichia pastoris expression system [27]. Although
several Ser and Thr residues in the ChuaHNL structure
are exposed to the solvent and there is only one predicted
O-glycosylation site [5], the electron density of O-linked
glycosylation site was not observed.
ꢀ
2.1 A resolution at an in-house X-ray source (Table 1).
The crystal belonged to P6122, and there was one
molecule in the asymmetric unit (Table 1 and Fig. 1A).
The amino acid residues were numbered from the N-
terminal Leu residue of the mature ChuaHNL, whose
signal peptide was removed. The electron density maps
of Leu1 at the N-terminal end and of the Tyr162 at the
C-terminal end of mature ChuaHNL were clearly visi-
ble. ChuaHNL contains two a-helices, three 310-helices,
and eight b-strands (Fig. 1 and 2B). The eight b-
strands form an antiparallel b-barrel comprising the
central active site cavity (Fig. 1B).
The homodimer structure was generated by the crystal-
lographic symmetry (Fig 1B). The buried dimer interface
Ligands bound in the active site of ChuaHNL
Acetate-bound form
2
area was 1,717 A corresponding to 20 % of the surface
ꢀ
area in the monomer structure. In the dimer interfaces,
hydrogen bonds formed mainly among loops connecting
b1-b2, b2-b3, b3-b4, b4-b5, and b5-b6, and hydrophobic
The acetate-bound form of ChuaHNL structure was
ꢀ
determined at 1.5 A resolution. The acetate contained
The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies
3

3D structure of hydroxynitrile lyase from a millipede
F Motojima et al.
Table 1. Statistics for data collection and refinement
Data set
Acetate
***
Iodide-SAD
Ligand free
***
Cyanide ion
***
Thiocyanate
***
Iodoacetate
***
PDB code
Data Collection
Beam line
PF BL1A
R-AXIS VII
PF BL5A
PF BL5A
PF BL1A
PF BL1A
Space group
Cell dimensions
P6122
P6122
P6122
P6122
P6122
P6122
ꢀ
a = b, c (A)
58.25, 224.98
1.1
58.42,
226.41
1.54
58.38, 226.09
1.0
58.59, 226.23
1.0
58.10, 225.96
1.1
58.09, 225.46
1.1
ꢀ
Wavelength (A)
ꢀ
Resolution (A)
50.44-1.50 (1.58- 46.19-2.10 (2.21- 50.56-1.60 (1.69- 50.74-2.10 (2.21- 50.31-1.55 (1.61- 50.31-1.55 (1.63-
2.10)*
1.50)*
1.60)*
2.10)*
1.55)*
1.55)*
Rmerge
0.046 (0.501)*
26.5 (4.1)*
100.0 (100.0)*
10.4 (10.8)*
0.117 (0.657)*
27.3 (5.0)*
100.0 (100.0)*
19.1 (15.8)*
0.039 (0.229)*
36.1 (9.5)*
99.9 (100.0)*
11.3 (11.6)*
0.116 (0.598)*
18.8 (5.3)*
100.0 (100.0)*
19.5 (20.5)*
0.049 (0.525)*
34.7 (6.1)*
99.9 (99.6)*
18.7 (16.9)*
0.066 (0.624)*
25.1 (4.2)*
100 (100)*
17.9 (16.6)*
I/ r(I)
Completeness (%)
Redundancy
Phasing
Anomalous
100 (100)*
completeness (%)
Anomalous multiplicity
FOM after SHELX
Refinement
10.8 (8.5)*
0.63
No. reflections
35572
29697
13571
32225
32191
R
work/Rfree
0.174/0.187
0.159/0.185
0.191/0.233
0.171/0.191
0.165/0.179
Average B-factor/Number of atoms
Protein
16.8/1303
35.6/32
18.5/1314
41.4/28
31.5/1287
84.6/38
18.1/1305
39.0/30
17.6/1310
43.0/33
Ligands
Waters
33.2/305
34.7/297
39.6/141
33.3/227
31.8/231
R.m.s. deviations
ꢀ
Bond lengths (A)
0.007
1.25
0.007
1.30
0.014
1.67
0.007
1.30
0.007
1.28
Bond angles (°)
*Values in parentheses are for the highest-resolution shell. R.m.s. deviations: root-mean-square deviations.
in the reservoir solution was observed in the active
site. The carboxylic group’s oxygen forms salt bridges
with R38 and Y40 (Fig. 3A).
electrostatic interaction between the negatively charged
carbon and R38. The nitrogen atom of the cyanide ion
formed a hydrogen bond with Y40-Og at the distance
of 3.3 A. The negatively charged carbon of cyanide
ion electrostatically interacts with R38-Ng1 at the dis-
ꢀ
tance of 4.2 A.
ꢀ
Ligand-free form
The ligand-free form of ChuaHNL structure was
ꢀ
determined at 1.6 A resolution. To remove the acetate
Inhibitor-bound form
bound in the active site, the crystal was soaked in the
solution containing bis-tris propane-citric acid buffer
as described in the Materials and Methods. Three
water molecules were observed in the active site
(Fig. 3B). They formed hydrogen bonds with R38,
Y103, and K117.
Our group had previously reported that iodoacetate
and thiocyanate inhibited the (R)-MAN synthesis
catalyzed by ChuaHNL [5]. We determined the
structures of the complexes formed with iodoacetate
ꢀ
and thiocyanate at 1.55 A resolution each. In the
complex structure with iodoacetate, the oxygen of
the carboxylic acid moiety formed salt bridges with
R38-N1g and -N2g with bond lengths of 3.1 and
Cyanide ion-bound form
ꢀ
The crystal structure of ChuaHNL complexed with a
ꢀ
cyanide ion was determined at 2.1 A resolution
3.0 A, respectively, and with K117-Nꢀ with bond
ꢀ
length of 3.1 A (Fig. 3D). The iodine atom of iodoac-
(Fig. 3C). The orientation of the cyanide ion was
determined by the temperature factor and a possible
etate may form a weak p-interaction with F67 with
bond length of 4.3 A. A strong anomalous difference
ꢀ
4
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F Motojima et al.
3D structure of hydroxynitrile lyase from a millipede
A
L2-3
L5-6
L2-3
C
1
1
N99
D19
N121
N21
5
6
5
6
Q42
1
1
2
L5-6
4
R45
W46
7
7
F159
N
N
8
8
2
4
1
1
2
3
3
3
2
C158
Y162
T60
4
2
C80
6
I62
C
C
3
3
H156
H81
Q61
NAG2
B
35-152
80-158
104-118
N
C
NAG1
3-108
104-118
3-108
NAG1
N
C
80-158
35-152
NAG2
Fig. 1. Overall structure of ChuaHNL. (A) The stereo view of the monomeric structure of ChuaHNL. The helices (a1-2 for a-helices and g1-3
for 310-helices), the b-strands (b1-8), and the loop structures are colored in magenta, green, and gray, respectively. ‘N’ and ‘C’ symbols
indicate the N- and C-terminal ends of ChuaHNL, respectively. (B) The dimer structure of ChuaHNL. One chain is colored in green and the
other is colored in blue. Disulfide bonds are labeled with the residue number and are shown in
a sphere representation. N-
acetylglucosamine moieties connecting to N109 and N123 are labeled as NAG1 and NAG2, respectively. N109, N123, and sugar moieties
are shown via a stick representation. (C) The dimer interface of ChuaHNL. The disulfide bonds, hydrogen bonds, and salt bridges in the
interface are colored in yellow, magenta, and cyan, respectively. The interface residues and b-strands are labeled. The protein structures
were designed using the program PyMOL [61]
map between F67 and R38 implies the presence of an
iodine atom.
and nitrile group of (R)-MAN interact with hydrophi-
lic residues of ChuaHNL at the bottom of the active
site. The benzene group of (R)-MAN is surrounded by
many hydrophobic and aromatic residues (such as I11,
F17, F25, A54, F67, A75, L77, W88, F90, A105, and
A119). The deep hydrophobic cavity might facilitate
the acceptance of various bulky cyanohydrins as
observed previously [5].
In the complex structure with thiocyanate, two alter-
nate binding patterns were observed. The orientation
of the two thiocyanate molecules in the crystal struc-
ture was determined by the lower B-factor values after
the structure refinement. In one binding model
(Fig. 3E, SCN1), the sulfur atom of thiocyanate
formed a hydrogen bond each with Y103-Og and
The interactions between hydrophilic residues and
(R)-MAN are shown in Fig. 4B. The hydroxyl group
of (R)-MAN forms hydrogen bonds with K117-Nꢀ and
ꢀ
R38-Ng1 with bond lengths of 3.0 and 3.2 A, respec-
tively. In the other binding model (Fig. 3E, SCN2),
the sulfur atom of thiocyanate formed salt bridges
with R38-Ng2 and K117-Nꢀ both with bond length of
ꢀ
R38-Ng1 both with bond length of 3.3 A. The nitrile
group forms hydrogen bonds with R38-Ng1, R38-Ng2,
ꢀ
ꢀ
3.1 A (Fig. 3E).
and Y103-Og with bond lengths of 3.4, 3.3, and 3.3 A,
respectively. D56 forms salt bridges with R38-Ng2, R38-
Nꢀ, and K117-Nꢀ. Y40-Og forms hydrogen bonds with
R38-Ng1 and Y103-Og. The bond lengths and orienta-
tions of the residues involved in the ligand binding were
almost identical in all five ChuaHNL structures.
The docking simulation of (R)-MAN
To model how ChuaHNL catalyzes the synthesis and
cleavage of cyanohydrins, we performed a docking
simulation of (R)-MAN into the active site of
ChuaHNL by MOE (Molecular Operating Environ-
ment version 2016.8, Montreal, Canada). The model
giving the most prominent affinity score (S = À5.44) is
shown in Fig. 4. The binding of (R)-MAN in the cav-
ity of active site is shown in Fig. 4A. The hydroxyl
Site-directed mutational analysis of ligand-
binding residues and kinetic studies
To prove the catalytic roles of the hydrophilic residues
that interact with (R)-MAN as indicated by the results
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5

3D structure of hydroxynitrile lyase from a millipede
F Motojima et al.
A
1
2
SCR1
4
3
1
3
SCR3
SCR1
SCR2
B
SCR2
SCR3
2
4
C
N
Fig. 2. Structural and sequential comparison of ChuaHNL with lipocalins. (A) The secondary structure-based multiple sequence alignment
among ChuaHNL and typical lipocalins, such as human retinol binding protein 4 (RET4_HUMAN, PDB ID: 1JYD, DALI Z-score: 13.2) [62,63],
rat epididymal-specific lipocalin 5 (LCN5_RAT, PDB ID: 1EPA, DALI Z-score: 12.5) [52], bilin binding protein (BBP_PIER, PDB ID: 1BBP, DALI
Z-score: 12.3) [64], and mouse major urinary protein 1 (MUP1_MOUSE, PDB ID: 2DM5, DALI Z-score: 10.2) [65]. Numbers refer to the
ChuaHNL sequence. The three major structurally conserved regions (SCRs 1-3) are indicated by rectangles. The secondary structure
elements are shown as follows: a-helices, medium squiggles with a symbols; 310-helices, squiggles with g symbols; b-strands, arrows with
b symbols; strict b-turns, TT letters. Identical residues are highlighted with red boxes and white texts. Similar groups are highlighted with
yellow boxes. Similar amino acids in each group are shown as bold characters. The pairs of cysteine residues forming disulfide bonds are
shown as green digits at the bottom of the alignment. The proposed catalytic residues are surrounded by blue boxes. The alignment was
done using PROMALS3D [66] and illustrated using ESPript 3.0 (http://espript.ibcp.fr) [67]. (B) The superposed structures of monomer
ChuaHNL (green) and human retinol binding protein 4 (PDB ID: 1JYD) are shown. The SCRs 1-3 of ChuaHNL are colored in yellow, orange,
and magenta, respectively. The N- and C-terminal ends of ChuaHNL polypeptide chain are labeled as N and C, respectively. The alignment
was done using PROMALS3D [67] and illustrated using ESPript 3.0 (http://espript.ibcp.fr) [67]. Superposition with secondary structure
matching was evaluated using superpose program of CCP4 suites [54]
6
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Federation of European Biochemical Societies

F Motojima et al.
3D structure of hydroxynitrile lyase from a millipede
B
A
K117
8
K117
8
D56
D56
K117
D56
2.9
3.4
2.8
3.4
2.8
2.9
R38
R38
7
7
3.1
R38
2.8
2.7
2.7
2
2
Y103
Y103
Y103
Y40
Y40
Y40
D
K117
E
C
K117
K117
D56
SCN2
3.1
3.0
3.1
D56
D56
3.1
3.2
4.2
3.3
R38
SCN1
R38
3.1
3.0
R38
F67
Y103
Y103
Y103
Y40
Y40
Y40
Fig. 3. The active sites of ChuaHNL complexed with various ligands. Amino acid residues in the active site and bound ligands are shown as
a stick representation in CPK color scheme (carbon atoms of protein and ligands are shown in green and cyan, respectively). Hydrogen
ꢀ
bonds and electrostatic interactions between amino acid residues and ligands are shown as dashed lines (orange) with bond length in A. rA-
weighted omit maps for ligands and water molecules are shown as a blue mesh representation and contoured at 3.0 r level except D (5.0
r). (A) The wall-eye stereo view of the active site bound to acetate. The surface of the active site cavity is shown as transparent gray
surface. The entrance to the active site is located at the bottom of the images. (B) The ligand-free form. Water molecules bound to the
active site are shown as a sphere representation. (C) The complex with cyanide ion. (D) The complex with iodoacetate. The anomalous
difference map is shown as a violet mesh representation and is contoured at 4.0 r level. (E) The complex with thiocyanate. Two alternate
forms of the complex with thiocyanate are shown and labeled (SCN1 and SCN2). The protein structures were designed using the program
PyMOL [61]
of the docking simulation, site-directed mutagenesis
was performed using the recombinant His-ChuaHNL
generated in P. pastoris by coexpression with protein
disulfide bond isomerase (PpPDI) as described in our
previous report [27]. The hydrophilic residues sur-
rounding (R)-MAN in the docking model (R38, Y103,
Y40, D56, and K117) were altered (Fig. 4A and 4B).
The Ala variant of R38 (R38A) was successfully
expressed and purified, but none of the other Ala vari-
ants were expressed in P. pastoris as confirmed by
western blotting analysis (data not shown). Alterna-
tively, analogous variants of His-ChuaHNL (Y40F,
D56E, Y103F, and K117R) were similarly prepared
and their kinetic parameters shown in the synthesis
and cleavage toward benzaldehyde and (R)-MAN,
respectively, were determined and compared with those
of the wild-type enzyme.
MAN cleavage (kcat = 142Æ0.85 sÀ1) activity. All of
the parameters obtained with mutations on the hydro-
philic residues surrounding (R)-MAN in the docking
model were decreased or impaired both in the synthe-
sis and cleavage activities. The cleavage activity of the
R38A variant was completely lost, indicating the criti-
cal importance of R38 which interacts with both the
hydroxyl and the nitrile groups of (R)-MAN in the
binding model. The Vmax, kcat, and kcat/Km of two
variants, Y103F and K117R, were significantly
decreased as compared with the wild-type, supporting
the above model that Y103 and K117 directly interact
with the nitrile and the hydroxyl groups, respectively,
of (R)-MAN. Some unexpected results of the Km val-
ues of the proposed mutations substitute with a bulky
aromatic amino acid side chain, Y40F and Y103F,
showed improved (R)-MAN binding affinities probably
because of hydrophobic p-p interactions between F40
or F103, generating and the (R)-MAN (Fig. 4B).
Another analogous variants, D56E and K117R, also
resulted in preferential recognition of (R)-MAN,
exhibiting the lower Km value than that of the wild-
The kinetic parameters of the cleavage and synthesis
of (R)-MAN by His-ChuaHNL and its variants are
shown in Table 2. The turnover number of His-
ChuaHNL
for
(R)-MAN
synthesis
(kcat = 2110Æ180 sÀ1) was higher than that of the (R)-
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Federation of European Biochemical Societies
7

3D structure of hydroxynitrile lyase from a millipede
F Motojima et al.
key residue that is involved in an acid/base catalysis in
the ChuaHNL reaction mechanism. The lowest Vmax,
A
F25
D56
A
K117
kcat, and kcat/Km toward benzaldehyde was calculated
to be 40 Æ 0.81 µmolÁminÀ1ÁmgÀ1, 14 Æ 0.28 s-1 and
W88
A105
À1
3.0 Æ 0.63 mM ÁsÀ1, respectively, with Y103F; these
values were 125-fold, 150-fold, and 143-fold lower
than those of wild-type His-ChuaHNL, respectively.
The Km of Y103F (4.90 Æ 1.1 mM) was almost identi-
cal to that of the wild-type (4.91 Æ 0.66 mM), suggest-
ing that the Y103 interacts with the cyanide ion, not
with benzaldehyde. The Vmax and kcat of mimic func-
tion of K117R variant was 403 Æ 45 µmolÁminÀ1ÁmgÀ1
and 141 Æ 15 sÀ1 which exhibited the highest activities
retaining synthesis activity among the variants,
whereas the Km value for benzaldehyde was eight times
higher than that the wild-type. Consequently, the
enzyme efficiency of this variant (kcat/Km = 3.6 Æ
R38
F90
Y40
À1
0.24 mM ÁsÀ1) was dramatically decreased, similar to
K117
B
that of Y103F variant. It is obvious in this variant
that K117 could have an effect on the accessibility and
affinity toward benzaldehyde.
8
3.3
3.3
2.8
(R)-MAN
Another analogous alteration at Y40F and D56E,
which do not look to interact with (R)-MAN directly,
the turnover numbers for (R)-MAN cleavage were
18 Æ 0.35 and 6 Æ 0.3 sÀ1, which retain 12.6% and
4.2% activity of the wild-type, respectively. These
results suggest that the mutations do not disrupt the
interaction with (R)-MAN and that D56 has a role in
the cleavage activity, whereas Y40 has an effect on the
synthesis activity, coordinating with benzaldehyde.
D56
2.9
2.8
3.3
3.4
3.3
2.9
3.0
Y103
R38
Y40
3
2
Discussion
Fig. 4. The docking simulation and mutational analysis of
ChuaHNL. (A) The surface representation of docked (R)-MAN in the
active site of ChuaHNL. (R)-MAN and the residues in the active
site are shown as a stick representation in CPK color scheme. The
carbon atoms of (R)-MAN are colored in yellow. The carbon atoms
of hydrophilic, negatively charged, positively charged, aliphatic, and
aromatic residues of ChuaHNL are colored in orange, pink cyan,
green, and purple, respectively. (B) The interactions between (R)-
MAN and the residues in the active site. Color scheme is the
same as in A. The hydrogen bonds interacting with (R)-MAN are
shown as orange dotted lines along with their lengths. The
hydrogen bonds and salt bridges between the residues are shown
as gray dotted lines. The docking simulation was done and the
figures drawn by MOE (Molecular Operating Environment version
2016.8, Montreal, Canada)
We have proved in C. hualienensis that (R)-Man is
biosynthesized from L-Phe, catalyzed by several P450
enzymes characterized by expression in E. coli of their
cDNA cloned from mRNA [28]. We established the
molecular mechanisms of the synthesis of defense allo-
mones and shown that adult millipedes produce a mix-
ture of benzaldehyde, benzyl alcohol, benzoylcyanide,
mandelonitrile, and benzoic acid [29,30]. They employ
various mechanisms to defend themselves using
enzyme-mediated secretions such as HCN from (R)-
MAN by HNL [5] and benzoyl cyanide and hydrogen
peroxide from (R)-MAN using a new enzyme mande-
lonitrile oxidase [31]. Benzoyl cyanide further accepts
one more molecule of (R)-MAN to form an ester (R)-
MAN benzoate by a nonenzymatic Schotten–Bauman
reaction releasing one molecule of cyanide [32].
type, possibly due to an improvement of the interac-
tion in the bond length and/or the favorable orienta-
tion of (R)-MAN.
Since these arthropods comprise highly new and
potential enzymes, we are interested in precisely study-
ing them from isolation and molecular characterization
of these valuable biocatalysts to the structural
In the synthesis reaction, the R38A variant also
showed no activity, indicating that R38 would be a
8
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Federation of European Biochemical Societies

F Motojima et al.
3D structure of hydroxynitrile lyase from a millipede
Table 2. Kinetic parameters of wild-type and mutants His-ChuaHNL
Cleavage reactionb (R-MAN as substrate)
Synthesis reactionc (Benzaldehyde as substrate)
Vmax
(µmol min-1 mg-1)
kcat
(s-1)
Km
kcat/ Km
(mM-1 s-1)
Vmax
(µmol min-1 mg-1)
kcat
(s-1)
Km
kcat/Km
(mM-1 s-1)
Enzyme*,a
(mM)
(mM)
Wild-type
R38A
406 Æ 2.11
n.d.
142 Æ 0.85
n.d.
8.7 Æ 0.40
n.d.
16.4 Æ 0.88
n.d.
5030 Æ 517
n.d.
2110 Æ 180
n.d.
4.9 Æ 0.6
n.d.
430 Æ 23
n.d.
Y40F
54 Æ 0.93
18 Æ 0.88
4 Æ 0.42
1.3 Æ 0.81
18 Æ 0.35
6 Æ 0.30
1.4 Æ 0.14
0.44 Æ 0.1
0.9 Æ 0.04
1.3 Æ 0.06
1.0 Æ 0.22
2.93 Æ 0.5
20.4 Æ 0.56
4.8 Æ 0.01
1.4 Æ 0.15
0.15 Æ 0.03
190 Æ 16
292 Æ 2.03
40 Æ 0.81
403 Æ 45
66 Æ 5.6
102 Æ 0.71
14 Æ 0.28
141 Æ 15
1.6 Æ 0.26
7.1 Æ 1.1
4.9 Æ 1.1
40 Æ 6.6
40 Æ 2.6
14.6 Æ 2.3
3.0 Æ 0.63
3.6 Æ 0.24
D56E
Y103F
K117R
aHis-ChuaHNL was prepared and purified by using a recombinant ChuaHNL in P. pastoris.; bThe kinetic parameters were obtained by moni-
toring cleavage of (R)-MAN by a UV-vis spectrometer at 280 nm.; cThe kinetic parameters were obtained by monitoring synthesis of (R)-
MAN by an HPLC at 254 nm.
determination and mechanism of function. It is already
known that the cleavage of cyanohydrins by other
HNLs is initiated by the deprotonation of the hydro-
xyl group by a general base [33]. As shown in Fig. 5,
the architectures of the active sites of several HNLs
hitherto known are largely different. A His residue is
commonly used as a general base in the HNLs belong-
ing to GMC oxidoreductase (Fig. 5A) [34,35], a/b-hy-
drolases (Fig. 5B, 5C) [36,37], dimeric a + b barrel
folds (Fig. 5D) [18], and cupin superfamily [3,38]. In
the a/b-hydrolase HNL family, Ser deprotonated by
His is used as a general base (Fig. 5B) [39]. In the Bet
v1 superfamily HNL (DtHNL), Tyr deprotonated by a
water bridges between the OH-group of Tyr and the
guanidinium group of Arg (Fig. 5E), which is an unu-
sual case among the known HNLs [9]. In the case of
ChuaHNL, the hydroxyl group of (R)-MAN interacts
with R38 and K117. Considering with a lower pKa of
Lys than Arg (10.5 and 12.5, respectively), K117
would act as a base in the cleavage reaction of
cyanohydrins. The deprotonated state of Lys is unsta-
ble due to its high pKa, this pKa should be perturbed
and decreased to the reactive pH 4–5 of ChuaHNL
(Fig. 5F). The hydrophobic environment of the active
site could stabilize the deprotonated state of K117,
resulting in the pKa decrease. It has been reported that
the pKa of Lys is decreased in a hydrophobic environ-
ment of various enzymes by excluding water molecules
which donate protons and Lys can act as a base which
abstracts a proton from substrates [40,41]. As observed
in several enzymes, it is possible that R38 could act as
a base [42]. The weak cleavage activity of K117R vari-
ant (kcat = 0.44 Æ 0.1 sÀ1) suggests that Arg may act
as a weak base. The proton abstracted by K117 is
donated to the released cyanide ion to produce a
hydrogen cyanide. However, the crystal structure of
the complex formed with a cyanide ion indicated that
the cyanide ion interacts with Y40 and R38 (Fig. 3C).
Therefore, R38 acts the proton donor for the produced
cyanide ion to form hydrogen cyanide (HCN). This
implies that in the catalytic pocket, hydrogen of R38 is
more prone to dissociate than HCN.
The interaction between the nitrile group and the
positively charged residues is found in other HNLs,
such as His and FAD in GMC oxidoreductase family,
Lys in a/b-hydrolase family, and His in cupin super-
family. In ChuaHNL, the nitrile group of cyanohydrin
interacts with R38 and Y103. Mutation in these resi-
dues caused enzyme inactivation, indicating the impor-
tance of the alignment of the nitrile group during the
enzymatic reaction.
From the crystal structures, docking simulation,
mutational analysis, and kinetic studies, we propose
the catalytic mechanism of ChuaHNL (Fig. 6). By the
desolvation effect in the hydrophobic active site, the
deprotonated state of K117 and the protonated state
of D56 are stabilized. As a result, the proton is shared
by K117 and D56 (Fig. 6A, the resting state). Upon
the binding of a cyanohydrin, K117 abstracts a proton
from cyanohydrin and an electron is received by the
carbon atom of the cyanide group (Fig. 6B). The pKa
of R38 is decreased to less than that of cyanide ion
(pKa: 10.5) by the desolvation effect in the hydropho-
bic active site. The released cyanide ion abstracts a
proton from R38 and a hydrogen cyanide is produced
(Fig. 6D). Aldehyde and cyanide ions are released
from the active site. The proton bound on D56 is
transferred to R38 and the active site is returned to
the resting state (Fig. 6D). Thus, in the proposed cat-
alytic reaction, K117 acts as a general base and R38
acts as a general acid.
During the synthesis reaction of cyanohydrins, the
reaction could occur as suggested for other HNLs.
The proton of the bound hydrogen cyanide was
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9

3D structure of hydroxynitrile lyase from a millipede
F Motojima et al.
abstracted by R38 and the negatively charged carbon
of cyanide ion acts as a nucleophile and attacks the
carbon of the aldehyde group of the substrate
(Fig. 6C). The hydrogen bound on K117 was
abstracted to the aldehyde group of the substrate con-
comitantly with the electron binding to the hydrogen
(Fig. 6B). The synthesized cyanohydrin is released out
of the active site of ChuaHNL (Fig. 6A). Therefore,
ChuaHNL utilizes a general acid/base catalysis mecha-
nism during the cleavage and synthesis reactions.
The lipocalin protein family is known as a large
group of small proteins distributed among three king-
dom of life, with the molecular masses mostly of 18 to
20 kDa, and their primary sequence diversity is large
[26]. Lakshmi et al report that the sequence identities
of over 80% of typical lipocalins are in the range as
A
B
C
D
E
F
Fig. 5. A general base catalysis model of HNLs. (A) Structure and proposed mechanism of GMC oxidoreductase (PaHNL). His residue acts
as a general base. (B) Structure and proposed mechanism of a/b-hydrolase (S-HbHNL). Ser deprotonated by His acts as a general base. (C)
Structure and proposed mechanism of a/b-hydrolase (R-AtHNL). Ser deprotonated by His acts as a general base. (D) Structure and proposed
mechanism of HNL with dimeric a + b barrels (R-PeHNL). His residue acts as a general base. (E) Structure and proposed mechanism of Bet
v1 superfamily HNL (R-DtHNL). Tyr deprotonated by a water bridging between the guanidinium group of Arg acts as a general base. (F)
Structure and proposed mechanism of ChuaHNL. The hydroxyl group of (R)-MAN interacts with R38 and K117. Lys acts as a general base
in the cleavage reaction of cyanohydrins. In all of the models, the first step of the catalysis is deprotonation from the hydroxyl group of
cyanohydrins. Except DtHNL and ChuaHNL, His residue acts as a base to protrude hydrogen cyanide from mandelonitrile, whereas DtHNL
and ChuaHNL use Tyr and Lys, respectively, as a general base. The negatively charged nitrile groups interact with basic residue except
PeHNL. PaHNL; Prunus amygdalus. HbHNL; Hevea brasiliensis. AtHNL; Arabidopsis thaliana. PeHNL; Passiflora edulis. DtHNL; Davallia
tyermannii. The protein structures were designed using the program PyMOL [61]
10
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Federation of European Biochemical Societies

F Motojima et al.
3D structure of hydroxynitrile lyase from a millipede
low as 1–20% [43]. On the other hand, their three-di-
mensional structures are highly conserved. It is
accepted that the 3D-structures are conserved better
than the amino acid sequences, which have been
traditionally used for sequence-based phylogenetic
approaches. They have several disulfide bonds. They
form a single eight-stranded hydrogen-bonded antipar-
allel b-barrel, which forms a binding site. Some of
them have abilities to bind small hydrophobic mole-
cules. They exhibit functional diversity, with roles in
transport of retinol, olfaction, cryptic coloration, and
pheromone. They also appear to function in
immunomodulation and the regulation of cell
homoeostasis. Violaxanthin de-epoxidase and zeaxan-
thin epoxidase of plant origins are grouped as lipoca-
lins from their primary structures [68]. Prostaglandin
D synthase (EC5.3.99.2) is one of a few characterized
enzymes belonging to the lipocalin family [26,44-47].
The enzyme catalyzes the isomerization of 9,11-en-
doperoxide group of prostaglandin precursor to pros-
taglandin D2. Recently, it was reported that the
protein has dual functional roles as the enzyme and as
an extracellular transporter for diverse lipophilic com-
pounds in the cerebrospinal fluid [48]. So, in our study,
the elucidation of the structure and function of
ChuaHNL and the future study seeking for ancestral
protein is of much interest based on its genome struc-
ture. Many protein frames with similar primary struc-
tures to ChuaHNL are found in the genome of C.
hualienensis (unpublished results), although these pro-
teins have no identity among database. Thus, it is indi-
cated that ChuaHNL might have an ancestral protein
which is functioning in the transport or olfactory
responses on low molecular weight compounds in the
arthropod.
In our previous study, ChuaHNL was found to be
synthesized and localized in the defensive secretory
gland(s) in the paraterga of the millipede by a histo-
chemical localization experiments [5,49]. The millipede
has a storage chamber of neat (R)-MAN synthesized
by the inner cells of the chamber, upstream of the way
out of the products HCN gas and benzaldehyde
through the hole called ozopore. It is noteworthy that
(R)-MAN is pooled neat in the storage chamber with-
out glycosylation, as we could not detect the glycosy-
lated (R)-MAN in the millipede [28]. In the next
chamber connected to the reaction chamber,
ChuaHNL is specifically biosynthesized and exuded at
the ozopore to outside through a duct of the ozopore
from the reaction chamber. Thus, because the neat
substrate would be supplied in a higher concentration
from the storage chamber to the next chamber
A
D
N
O
O
2
N
2
O
O
1
N
1
N
B
C
Fig. 6. The proposed catalytic cycle of ChuaHNL. (A) The resting state. The deprotonated state of Lys117 is stabilized by the desolvation
effect in the hydrophobic active site. The residues are labeled and colored in cyan. (B) The binding of cyanohydrin in the active site. The
bound cyanohydrin is colored in red. Lys117 forms a hydrogen bond with the cyanohydrin and abstracts a proton from the cyanohydrin. The
electron released from the hydrogen is received by the carbon atom of the nitrile group. (C) The formation of hydrogen cyanide. The
released cyanide ion abstracts a proton from Arg38. Subsequently, aldehyde and cyanide ions are released out of the active site. (D) The
recovering state. The proton bound on Asp56 is transferred to Arg38 and the active site is returned to the resting state
The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies
11

3D structure of hydroxynitrile lyase from a millipede
F Motojima et al.
containing HNL and the product HCN gas and ben-
zaldehyde are pushed out instantly through the ozo-
pore to outside the paraterga by the action of the
animal, higher Km value (8.7 mM) for (R)-MAN and
the extremely high specific activity for (R)-MAN cleav-
age (406 UÁmgÀ1) shown by ChuaHNL are quite rea-
sonable, not like most of other plant HNLs which
usually act in lower substrate concentrations after the
cyanogenic glycosides are hydrolyzed by b-glucosidase
reaction [50]. The Vmax values of the HNLs from plant
origins are compared: they usually show less than 100
UÁmgÀ1 in the degradation of MAN and sometime
more than 100 UÁmgÀ1 in MAN synthesis. Km values
for MAN and benzaldehyde are around 1.0 mM and
10 mM or less, respectively [2,23].
catalyzes this reaction while it evolutionally forms a
new scaffold.
Materials and Methods
Materials and chemicals
The invasive millipedes Chamberlinius hualienesis were man-
ually collected in the Japanese cedar forest located in
Kyushu, Japan, and were stored at À80°C until use for
protein purification. Benzaldehyde (redistilled, 99.5%), (R)-
MAN, and (rac)-MAN were purchased from Sigma-Aldrich
(St. Louis, MO, USA). All chemicals used for protein crys-
tallization were purchased from Hampton Research (Aliso
Viejo, CA, USA). Other chemicals were used in the experi-
ments were purchased from chemical sources and used
without further purification.
A phylogenetic analysis was performed for the
ChuaHNL, with the four similar structures obtained
by DALI search and 42 kinds of typical lipocalins hav-
ing diverse primary sequences and the 3D structures
[43]. Fig. S2 shows that the primary structure of
ChuaHNL is close to rat epididymal retinoic acid
binding protein (PDB ID: 1EPA) [52,63]. In Fig. S3,
structural superimposition and a comparison were
made on the size and shape of the binding cavity of
ChuaHNL with the lipocalin family proteins, having
high Z-scores by DALI search, which are shown in
Fig. 2: human retinol binding protein 4 (RBP4) and
rat epididymal-specific retinoic acid binding protein,
lipocalin 5 (LCN_RAT). In Fig. S4, structural super-
imposition and comparison of catalytic pockets in
ChuaHNL and prostaglandin D synthase were made.
Although the 3D structure of ChuaHNL showed simi-
larities with these proteins, the direction and the size
of the entrances for the substrates, and the depths of
the cavities are quite different, show in the unique
structure of ChuaHNL.
Enzyme assays
The enzyme activities for (R)-MAN synthesis and MAN
cleavage were assayed by HPLC and spectrophotometric
methods, respectively. The HNL activity during (R)-MAN
synthesis was measured as reported previously [5]. In brief,
the reaction (total volume = 1 mL) was initiated by adding
potassium cyanide (KCN; 100 mM) in citrate buffer
(400 mM, pH 4.2) containing benzaldehyde (50 mM; 40 µL
of 1250 mM benzaldehyde dissolved in dimethyl sulfoxide
(DMSO)) and enzyme solution (0.5-5 UÁmLÀ1). The mixture
was incubated at 22 ˚C for 5 min, and then, aliquots (100
µL) of reactant were mixed with 9-fold volume of n-hex-
ane:2-propanol (85:15) mixture. Finally, the organic phase
was analyzed using an HPLC instrument (UFLC Promi-
nence Liquid Chromatography LC-20AD, Shimadzu,
Kyoto, Japan) equipped with a chiral column (CHIRAL-
CEL OJ-H, Daicel, Osaka, Japan) (i.d. 4.6 mm 9 250 mm
length, 5 lm particle size). One unit of synthesis activity was
defined as the amount of the enzyme that synthesizes 1 lmol
of (R)-MAN from benzaldehyde and KCN per min.
The significance of the current research is that the
enzyme shows a totally novel primary structure as
well as a new 3D structure, and the new key resi-
dues in the catalysis, as compared with the known
HNLs. The addition of our arthropod HNL with
the high similarity of the three-dimensional structure
to the lipocalin family, well-studied proteins with
variety of functions in mammals, indicates that we
are expanding the evolutionary relationship of HNLs
to a new family of proteins since these enzymes
would be good examples of convergent evolution.
HNLs are from various protein families which cat-
alyze the same reaction in nature. One of the most
important HNLs is originated from almond (R-
PaHNL) which looks like an oxidase activity, as evi-
denced also by its 3D structure [14]. Therefore, it is
noteworthy that the new HNL from C. hualienensis
For MAN cleavage activity assay, the reaction was
started by adding enzyme sample to a reaction mixture
(1 mL) consisting of racemic mandelonitrile or (R)-MAN
(2 mM) in citrate buffer (100 mM, pH 5.0). The reaction
velocity of benzaldehyde formation was followed by moni-
toring absorbance at 280 nm for one min (extinction coeffi-
cient of benzaldehyde = 1,352 L mol-1 cm-1) using a UV-
2600 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan).
Natural ChuaHNL preparation and
crystallography
The native ChuaHNL protein was purified by the protocol
reported previously [5]. In brief, first, a homogenate of mil-
lipedes solubilized in buffer-A (20 mM potassium
12
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Federation of European Biochemical Societies

F Motojima et al.
3D structure of hydroxynitrile lyase from a millipede
phosphate, pH 7.0), was fractionated by an ammonium sul-
fate precipitation procedure. Next, the native ChuaHNL
protein was purified using TOYOPEARL Butyl-650M
(Tosoh, Tokyo, Japan), TOYOPEARL DEAE-650M
(Tosoh, Tokyo, Japan), Q Sepharose FF (GE Healthcare,
Chicago, IL, USA), and Superdex 75 10/300 GL (GE
Healthcare, Chicago, IL, USA). Finally, the active fractions
were collected and subjected to sodium dodecyl sulfate/
polyacrylamide gel electrophoresis (SDS–PAGE) [51] to
evaluate their purity. For protein crystallization, the buffer
was replaced by 50 mM citrate buffer (pH 5.0) and then
was concentrated up to 10 mg ml-1 by using an Amicon
Ultra Centrifugal Filter Unites NMWL, 10 kDa (Merck
Millipore, Billerica, MA, USA). Protein concentration was
measured using a Quick Start Protein Assay (Bio-Rad Lab-
oratories, Hercules, CA, USA) using bovine serum albumin
(Sigma-Aldrich, St. Louis, MO, USA) as a standard.
[56] and refined using either REFMAC5 [57] or PHENIX
[58] programs.
Structural Analysis and Docking Simulation
All structures reported in this paper were validated using
MolProbity [59], and there was no residue in disallowed
regions of the Ramachandran plot. Statistics for phase
determination and those for data collections and structure
refinements are listed in Table 1. The structural analysis
was performed using PISA [60]. The protein structures were
designed using the program PyMOL (http://www.pymol.
org) [61]. (R)-MAN was docked with ChuaHNL by using
the Molecular Operating Environment program (MOE; ver-
sion 2016.8, Montreal, Canada).
Microbial strains, media, and culture condition
First, the crystallization conditions were screened using
CrystalScreen I and II (Hampton Research) and bipyrami-
dal crystals were obtained using the following conditions:
0.2 M ammonium sulfate, 0.1 M sodium acetate (pH 4.6),
and 30 % (w/v) polyethylene glycol (PEG) monomethyl
ether 2,000. Finally, the best crystals were obtained using a
hanging drop vapor diffusion method at 20 ˚C for 3 days
in the following conditions: 0.2 M ammonium sulfate,
0.1 M sodium acetate (pH 5.0), 28-32 % (w/v) PEG mono-
methyl ether 2,000, and 0.3 M NDSB-195.
Escherichia coli DH5a (Takara Bio, Otsu, Japan) was inoc-
ulated in Luria–Bertani (LB) medium (1% tryptone, 0.5%
yeast extract, and 0.5% NaCl) containing zeocin
(25 µgÁmLÀ1) for plasmid propagation. P. pastoris cells
harboring PpPDI/GS115 was used as host for the recombi-
nant His-ChuaHNL expression as described previously [27].
The yeast strain was precultured in a buffered minimal
glycerol (BMG) medium (1.34 % yeast nitrogen base with-
out amino acids, 4 9 10-5 % biotin, 100 mM potassium
phosphate buffer (pH 7.0), and 1 % glycerol) and then the
yeast cells were transferred to induction BMM medium
(BMG in which 1 % glycerol was replaced by 1 % metha-
nol). The culture was grown at 28 ˚C under aerobic condi-
tions with reciprocal shaking (150 rpm), and the growth of
the yeast was monitored by measuring the optical density
at 600 nm wavelength.
For the phase determination, a native ChuaHNL crystal
was soaked in the reservoir solution containing 0.5 M
sodium iodide at 20 ˚C for 30 min prior to freezing and
data collection. However, for the native and recombinant
structures complexed with acetate, the obtained crystals
were directly used for the data collection. For the ligand-
free form, a native protein crystal was soaked in soaking
solution-1 (32 % (w/v) PEG monomethyl ether 2,000,
0.3 M NDSB-195, 50 mM bis-tris-propane, and 50 mM
citric acid; pH 4.5) at 20 ˚C for 20 min to remove acetate.
For complexes with thiocyanate, iodoacetate, and cyanide
ion, crystals of the ligand-free form were soaked in soaking
solution-1 containing either 80 mM potassium thiocyanate,
10 mM sodium iodoacetate, or 0.5 M potassium cyanide,
respectively. All crystals were cryoprotected with perfluo-
roether and the data sets were collected at 100 K under the
nitrogen stream. The X-ray diffraction data for single
anomalous dispersion (SAD) phasing were collected using
an in-house X-ray generator and an imaging plate (Micro-
MAX-007 and R-AXIS VII, Rigaku, Tokyo, Japan). Other
data were collected using a silicon pixel detector (Pilatus
Construction of His-ChuaHNL variants
for expression in P. pastoris
The site-directed mutagenesis was performed using a
Quick-Change Lightning Site-Directed Mutagenesis
Kit (Agilent Technologies, Santa Clara, CA, USA).
The primer pairs are listed in Table S1 and pPICZaA-
His-ori-ChuaHNL vector [27] was used as template.
The PCR products were treated with DpnI (TaKaRa)
at 37 ˚C for 1 h and then transformed to E. coli
DH5a strain and selected on a low-salt LB agar plate
with Zeocin (25 µgÁmLÀ1). The mutant plasmids were
isolated by a plasmid miniprep kit (Sigma-Aldrich),
and the amino acid exchanges were confirmed by
DNA sequencing analysis. The constructed vector
pPICZaA-His-ori-ChuaHNL mutant was then lin-
earized by digesting with SacI and electroporated into
P. pastoris PpPDI/GS115, according to our previous
report [27].
2M-F, DECTRIS, Baden-Daettwil, Switzerland) and
a
CCD detector (Quantum 315r, Area Detector Systems,
Poway, CA, USA) at Photon Factory beamline BL-1A and
BL-5A (Tsukuba, Japan).
All data sets were integrated using XDS [53] and scaled
using SCALA [54]. The initial phase was determined using
SHELX suite [55]. All models were corrected using COOT
The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies
13

3D structure of hydroxynitrile lyase from a millipede
F Motojima et al.
Science) from Japan Agency for Medical Research and
Development (AMED).
Expression and purification of His-
ChuaHNL in P. pastoris system
The coexpression strain, P. pastoris ori-His-
ChuaHNL/PpPDI/GS115 mutant, was expressed and
purified as described previously [27]. Initially, after six
days of an induction BMM medium cultivation, the
pH of the supernatant was adjusted to 7.5 by addition
of 2 M sodium hydroxide. Next, the His-tagged pro-
tein was purified using Ni Sepharose 6 fast flow col-
umn (GE Healthcare, Chicago, IL, USA), Mono Q 5/
50 GL (GE Healthcare, Chicago, IL, USA). Finally,
the purified His-ChuaHNL mutants were dialyzed into
20 mM KPB buffer (pH 7.0) and analyzed the purity
by SDS–PAGE.
Author contributions
FM, AI, and YA planned experiments; FM, AI, AN,
ZZ, MD, SS, TY, and SN performed experiments;
FM, AI, AN, TY, and SN analyzed data; and FM,
AI, AN, and YA wrote the paper.
Conflicts of interest
The authors declare no conflict of interest.
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This work was supported by ERATO (Exploratory
Research for Advanced Technology Program), Asano
Active Enzyme Molecule Project of Japan Science and
Technology Agency (Grant No. JPMJER1102). This
research was also supported by a grant-in-aid for Sci-
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Federation of European Biochemical Societies
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