ArticlePDF Available

A sacrificial millipede altruistically protects its swarm using a drone blood enzyme, mandelonitrile oxidase

Authors:

Abstract and Figures

Soldiers of some eusocial insects exhibit an altruistic self-destructive defense behavior in emergency situations when attacked by large enemies. The swarm-forming invasive millipede, Chamberlinius hualienensis, which is not classified as eusocial animal, exudes irritant chemicals such as benzoyl cyanide as a defensive secretion. Although it has been thought that this defensive chemical was converted from mandelonitrile, identification of the biocatalyst has remained unidentified for 40 years. Here, we identify the novel blood enzyme, mandelonitrile oxidase (ChuaMOX), which stoichiometrically catalyzes oxygen consumption and synthesis of benzoyl cyanide and hydrogen peroxide from mandelonitrile. Interestingly the enzymatic activity is suppressed at a blood pH of 7, and the enzyme is segregated by membranes of defensive sacs from mandelonitrile which has a pH of 4.6, the optimum pH for ChuaMOX activity. In addition, strong body muscle contractions are necessary for de novo synthesis of benzoyl cyanide. We propose that, to protect its swarm, the sacrificial millipede also applies a self-destructive defense strategy—the endogenous rupturing of the defensive sacs to mix ChuaMOX and mandelonitrile at an optimum pH. Further study of defensive systems in primitive arthropods will pave the way to elucidate the evolution of altruistic defenses in the animal kingdom.
Content may be subject to copyright.
1
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
www.nature.com/scientificreports
A sacricial millipede altruistically
protects its swarm using a drone
blood enzyme, mandelonitrile
oxidase
Yuko Ishida1,2, Yasumasa Kuwahara1,2, Mohammad Dadashipour1,2, Atsutoshi Ina1,2,
Takuya Yamaguchi1,2, Masashi Morita1,2, Yayoi Ichiki1,2 & Yasuhisa Asano1,2
Soldiers of some eusocial insects exhibit an altruistic self-destructive defense behavior in emergency
situations when attacked by large enemies. The swarm-forming invasive millipede, Chamberlinius
hualienensis, which is not classied as eusocial animal, exudes irritant chemicals such as benzoyl
cyanide as a defensive secretion. Although it has been thought that this defensive chemical was
converted from mandelonitrile, identication of the biocatalyst has remained unidentied for 40 years.
Here, we identify the novel blood enzyme, mandelonitrile oxidase (ChuaMOX), which stoichiometrically
catalyzes oxygen consumption and synthesis of benzoyl cyanide and hydrogen peroxide from
mandelonitrile. Interestingly the enzymatic activity is suppressed at a blood pH of 7, and the enzyme is
segregated by membranes of defensive sacs from mandelonitrile which has a pH of 4.6, the optimum pH
for ChuaMOX activity. In addition, strong body muscle contractions are necessary for de novo synthesis
of benzoyl cyanide. We propose that, to protect its swarm, the sacricial millipede also applies a self-
destructive defense strategy—the endogenous rupturing of the defensive sacs to mix ChuaMOX and
mandelonitrile at an optimum pH. Further study of defensive systems in primitive arthropods will pave
the way to elucidate the evolution of altruistic defenses in the animal kingdom.
Swarm-forming animals have unique defense systems for protection. In eusocial insects that establish sophisti-
cated castes, such as ants, for example, the ant soldiers use defensive behaviors that involve releasing defensive
chemicals, biting with their mandibles, and stinging1. To address emergencies when attacked by large enemies,
some ants and termites have evolved “Kamikaze” or altruistic self-destructive defense behavior as an instantane-
ous defense2–4.
On the other hand, the swarm-forming primitive arthropods, millipedes, usually individually use irritant
chemicals to avoid attacks from omnivorous or carnivorous predators instead of establishing castes like the euso-
cial animals5,6. Mandelonitrile is a defensive chemical that is conserved among cyanogenic millipedes and is
also used as a starting material to produce benzaldehyde, hydrogen cyanide, and benzoyl cyanide as defensive
secretions. e aldoxime-nitrile pathway in the synthesis of mandelonitrile through phenylacetaldoxime and
phenylacetonitrile is widely observed in bacteria, cyanogenic plants, and millipedes7–10.
e invasive polydesmid millipede, Chamberlinius hualienensis Wang, derives from Taiwan11. e animal usu-
ally forms a large swarm in cedar forests (see Supplementary Fig. S1) and has been expanding its range through-
out southern Japan. When a cyanogenic millipede is attacked by a predator, it achieves a high blood pressure by
forming a tight defensive spiral and can then expel benzoyl cyanide as a defensive chemical5,6,12,13. is chemical
can disrupt the ant antennae functions, thus acting as an eective repellent. In addition, the reactive chemical
causes irritation of the nose, eyes, and mouth of vertebrates such as birds, lizards, and humans14. It has been pro-
posed that benzoyl cyanide is converted from mandelonitrile by dehydrogenation5. However, identication of
mandelonitrile dehydrogenase has remained unidentied for 40 years.
1Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa,
Imizu, Toyama 939-0398, Japan. 2Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu,
Toyama 939-0398, Japan. Correspondence and requests for materials should be addressed to Y.A. (email: asano@
pu-toyama.ac.jp)
received: 05 January 2016
accepted: 29 April 2016
Published: 06 June 2016
OPEN
www.nature.com/scientificreports/
2
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
Here, we identify the novel enzyme that catalyzes the synthesis of benzoyl cyanide from mandelonitrile. e
enzyme is actually classied as an oxidase. We characterize its enzymatic activity, physico-chemical properties,
and localization. Surprisingly, the substrate and the enzyme are not colocalized; they separately accumulate in
defensive sacs and in blood respectively. Synthesis of benzoyl cyanide likely occurs by, endogenously rupturing
the membranes of the defensive sacs using strong body muscle contractions during defensive behavior. We dis-
cuss this self-destructive defense of the millipede for protection of its swarm by synthesizing benzoyl cyanide
from mandelonitrile through mandelonitrile oxidase.
Results
Identication, purication, and characterization of mandelonitrile oxidase from the invasive
millipede. On the basis of a preliminary experiment using millipede homogenate as an enzyme source,
we detected the production not only of benzoyl cyanide but also of hydrogen peroxide from mandelonitrile.
To characterize the enzyme, we purified it using ion exchange and gel-filtration column chromatographies
(see Supplementary Table S1) and analyzed its physico-chemical properties and activities.
e puried enzyme was capable of synthesizing benzoyl cyanide from mandelonitrile in vitro (Fig.1a; see
Supplementary Fig. S2), the conditions (0.1 U of ChuaMOX, 1 μ mol of mandelonitrile, and 100 μ l of aqueous
buer) for which were estimated based on values from an animal extract and crude homogenate15. Placing
0.1 U and 0.01 U of the puried enzyme in 1 ml of 100 mM citrate buer with pH 5 containing 500 nmol of
Figure 1. Characterization of the puried ChuaMOX. (a) Synthesis of benzoyl cyanide from racemic
mandelonitrile. ChuaMOX is able to catalyze the synthesis of benzoyl cyanide at pH 5, and the retention times
for benzaldehyde, benzoyl cyanide, (R)-mandelonitrile, and (S)-mandelonitrile were 5.4 min, 6.1 min, 11.4 min,
and 14.3 min, respectively. Arrows indicate the peaks corresponding to benzaldehyde, benzoyl cyanide,
and racemic mandelonitrile. (b) Reaction of ChuaMOX. (c) Optimum pH. (d) pH stability. (e) Optimum
temperature. (f) Temperature stability. In panels (cf), the highest mean value of the activity at pH 4 in panel
c was dened as 100% to determine relative activity. Values are the means ± SD; n = 3. In panels (c,d), the
symbols , , and  indicate citrate, phosphate, Tris-HCl, and glycine-sodium hydroxide buers, respectively.
www.nature.com/scientificreports/
3
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
(R)-mandelonitrile at 25 °C for 1 min consumed 95.6 nmol and 7.30 nmol of oxygen and produced 106 nmol and
10.2 nmol of benzoyl cyanide and 100 nmol and 10 nmol of hydrogen peroxide, respectively. ese results indicate
that the enzyme stoichiometrically consumes oxygen and converts (R)-mandelonitrile into benzoyl cyanide and
hydrogen peroxide, (Fig.1b). e results suggest that the enzyme should be newly classied as mandelonitrile:ox-
ygen oxidoreductase (EC 1.1.3.-). us, we named this enzyme mandelonitrile oxidase (ChuaMOX).
e molecular mass of ChuaMOX was estimated to be 67,000 Da by SDS-PAGE analysis (see Supplementary
Fig. S3a) and 65,000 Da by gel-ltration. Periodic acid-Schi (PAS) staining determined that this enzyme was
glycosylated (see Supplementary Fig. S3b). UV-visible scanning detected three absorption maxima at 278 nm,
404 nm, and 469 nm (see Supplementary Fig. S4). in layer chromatography (TLC) identied the prosthetic
group as avin adenine dinucleotide (FAD) (see Supplementary Table S2).
e values of Km and Vmax of ChuaMOX for racemic mandelonitrile were calculated as 2.1 mM and 151.5 U/mg,
respectively. e optimum pH toward mandelonitrile lay within a range of pH 4 and pH 4.5, and the enzyme
also showed 80% activity at pH 3.5 and pH 5. Enzymatic activity decreased in conditions approaching neutral
pH, and it was lowest at a pH of 7 (Fig.1c). Aer one hour of incubation at 25 °C, 80% of the ChuaMOX activity
remained over a range of pH values between 3 and 10 (Fig.1d). e optimum temperature was 35 °C, and 90% of
the enzymatic activity was observed at 30 °C and 40 °C (Fig.1e). Aer one hour of incubation at pH 8, 80% of the
activity remained between 25 °C and 50 °C, and all activity was lost at 70 °C (Fig.1f). ese results suggest that
ChuaMOX is a stable enzyme similar to hydroxynitrile lyase, which was previously isolated from C. hualienensis
(ChuaHNL) and considered as a potential industrial biocatalyst for the synthesis of cyanohydrins8. e forma-
tion of hydrogen peroxide constituted 100%, 29%, and 22% of the ChuaMOX activity toward mandelonitrile,
(E)-2-hydroxy-4-phenylbut-3-enenitrile, and 2-(3-bromophenyl)-2-hydroxyacetonitrile, respectively, indicating
that this enzyme specically reacts with mandelonitrile (Table1). e enzyme required (R)-mandelonitrile as a
substrate rather than (S)-mandelonitrile (see Supplementary Table S3). Adding 1 mM potassium cyanide, sodium
azide, and 8-hydroxyquinoline resulted in 12.7%, 28.6%, and 56% relative activities of ChuaMOX, respectively,
but adding 1 mM ferric chloride enhanced the activity to 124.6% (see Supplementary Table S4).
Cloning of cDNA of ChuaMOX, prediction of its properties, and the enzyme and substrate local-
ization. To further characterize ChuaMOX, we cloned its cDNA. Based on protein sequencing, cDNA was
cloned using RNAseq data (BioProject ID, PRJDB3791) and gene-specic primers. e ORF consisted of 1,782 bp
encoding 593 amino acid residues including a 17 amino acid-long signal peptide, and the deduced amino acid
sequence also included 8 amino acid sequences determined by protein sequencing (accession number, LC036560;
see Supplementary Fig. S5). e predicted mature protein had a molecular mass of 62,818 Da and an isoelectric
point of 6.26. It also contained 6 predicted N-glycosylation sites (N57, N79, N94, N149, N406, and N491), and a
FAD binding motif (VXGXGXXGXXXA)16 in the T22-S62 region (see Supplementary Fig. S5), which agree with
the results of PAS staining, the UV-visible scanning, and the TLC analyses (see Supplementary Figs S3b and S4,
and Table S2). BLASTP showed that it had a 51% amino acid identity to the predicted glucose dehydrogenase
from the Florida carpenter ant, Camponotus oridanus (accession number, XP_011251213)17. However, phyloge-
netic analysis indicated that ChuaMOX did not belong to the cluster of glucose dehydrogenase and alcohol dehy-
drogenase (Fig.2). e enzymatic activity not to react toward D-glucose (Table1; see Supplementary Table S5)
and the phylogenetic tree (Fig.2) suggest that ChuaMOX is a distinctive enzyme stoichiometrically catalyzing
oxygen consumption and the conversion of mandelonitrile into benzoyl cyanide and hydrogen peroxide and that
it diers from glucose dehydrogenases and alcohol dehydrogenases.
ChuaMOX was expressed in the paraterga of the millipede (Fig.3a), which houses the storage and reaction
chambers related to the production of defensive secretions6. Because the storage and reaction chambers were
fragile in various preparations of tissue sections, we could not detect cytological expression of ChuaMOX. us,
we applied zymography to detect the enzyme activity of ChuaMOX from each experimental tissue. Intriguingly,
most of the enzyme was detected in the blood (0.1 animal equivalent) (Fig.3b), even though its pH of 7 is inap-
propriate for the expression of the enzymatic activity (Fig.1c; see Supplementary Fig. S6). A scanty amount of
the enzymatic activity was detected in the paraterga (1 animal equivalent), which may be derived from residual
blood in the tissue (Fig.3b). (R)-Mandelonitrile, a substrate of ChuaMOX, was detected from the paraterga by
HPLC analysis but benzoyl cyanide was not (Fig.3c). In addition, although defensive secretions such as ben-
zaldehyde, benzoyl cyanide, and benzoic acid were observed in the whole-body extract (Fig.3d), no candidate
cyanohydrins for ChuaMOX were detected from the blood by gas chromatography-mass spectrometry (GC/MS)
analysis (Fig.3e). ese results indicate that ChuaMOX is not only suppressed by the neutral pH of the blood, but
it is also segregated from its substrate, (R)-mandelonitrile, by tissue membranes of the storage and reaction cham-
bers. Furthermore, benzoyl cyanide is not stored in the defensive sacs but is newly synthesized during defensive
behavior. In other words, the expression of benzoyl cyanide synthesis by the defense system is unusual in terms
of the millipede’s physiology.
Body muscle contractions are essential for mixing ChuaMOX and (R)-mandelonitrile, causing
the production of benzoyl cyanide. To understand this enigma, we reexamined the synthesis of benzoyl
cyanide in the millipede. e animal discharges defensive secretions, the major components of which are man-
delonitrile, benzaldehyde, and benzoyl cyanide12,13. In the Japanese cedar forest, the animal is possibly attacked
by large predators such as wild birds (jungle crow, Corvus macrorhynchos; Eurasian jay, Garrulus glandarius;
dusky thrush, Turdus eunomus; and pale thrush, Turdus pallidus). ese birds usually show behaviors of 1) swal-
lowing in one gulp or 2) eating aer teasing with a beak. us, we mimicked such attacks for 1) as a non-treated
animal and 2) as a shaken animal, extracted the defensive secretion from each animal with an organic solvent
(n-hexane:2-propanol with a volume ratio of 85:15). We then analyzed the extract using an HPLC equipped with
a chiral column. e non-treated animal secreted benzaldehyde and benzoyl cyanide (Fig.4a), while the shaken
www.nature.com/scientificreports/
4
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
animal secreted benzaldehyde, benzoyl cyanide, and mandelonitrile (Fig.4b). ese results indicate that an active
millipede is able to produce benzoyl cyanide in both conditions 1) and 2).
In the n-hexane/2-propanol extraction, we reproducibly observed that both experimental animals writhed
in agony and exhibited strong body muscle contractions until death. It has been reported that a predator, the
larva of the phengodid beetle, Phengodes laticollis, presumably injects a muscle relaxant into its millipede prey,
Floridobolus penneri, before feeding to avoid the formation of a tight defensive spiral and the discharge of defen-
sive secretions18. us, we hypothesized that inhibition of the body muscle contractions presumably prevents the
production of benzoyl cyanide from the (R)-mandelonitrile. Aer anesthetizing using diethyl ether, the relaxed
millipede did not display its defensive form or muscle contractions. HPLC analysis failed to detect not only
benzaldehyde and mandelonitrile but also detected no benzoyl cyanide from the anesthetized animal (Fig.4c).
Furthermore, we sought to synthesize benzoyl cyanide by roughly snatching the paraterga from the weakly
anesthetized animals, which is expected to rupture defensive sac and mix residual blood containing ChuaMOX
and mandelonitrile. HPLC analysis detected benzaldehyde, mandelonitrile, and benzoyl cyanide (Fig.4d).
Substrate Structure Relative activity, %
Racemic mandelonitrile 100
(E)-2-Hydroxy-4-phenylbut-3-enenitrile 29
2-(3-Bromophenyl)-2-hydroxyacetonitrile 22
2-Hydroxy-2-(2-methoxyphenyl)acetonitrile 17
(3E,5E)-2-Hydroxydeca-3,5-dienenitrile 10
2-Hydroxy-2-(o-tolyl)acetonitrile 9
2-(4-Bromophenyl)-2-hydroxyacetonitrile 6
2-Hydroxy-2-(naphthalen-1-yl)acetonitrile 4
2-(2-Chlorophenyl)-2-hydroxyacetonitrile 3
2-Hydroxy-2-(4-methoxyphenyl)acetonitrile 3
2-(2-Bromophenyl)-2-hydroxyacetonitrile 2
2-Hydroxy-2-(naphthalen-2-yl)acetonitrile 2
(3E,5E)-2-Hydroxyocta-3,5-dienenitrile 1
Table 1. Substrate specicity of ChuaMOX. ChuaMOX activity toward test substrates was detected under
standard assay condition using ATBS. e relative activity was expressed as a percentage of the enzyme toward
mandelonitrile (0.1 U/ml). e enzyme activity toward other substrates listed in Table S5 was not detected.
www.nature.com/scientificreports/
5
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
These results suggest that synthesis of benzoyl cyanide requires strong body muscle contractions in the
tight defensive spiral position, presumably rupturing the defensive sacs and thus mixing ChuaMOX and
mandelonitrile.
Discussion
In this study, we isolate and localize ChuaMOX, which has remained unidentied for 40 years. It is stable in
a wide range of pH and temperature conditions and is a monomeric glycosylated oxidase containing FAD as
a prosthetic group. Furthermore, it stoichiometrically catalyzes the oxygen consumption and the synthesis of
benzoyl cyanide and hydrogen peroxide from (R)-mandelonitrile and is dierent from glucose dehydrogenase
and alcohol dehydrogenase. us, this enzyme is classied as mandelonitrile:oxygen oxidoreductase (EC 1.1.3.-).
e enzymatic activity is expressed in a range of pH 3.5 to pH 5, whereas it is lowest at a blood pH of 7. e blood
enzyme is segregated by membranes of the storage and reaction chambers from the substrate, (R)-mandelonitrile,
in a condition of pH 4.6 (see Supplementary Fig. S6). In v ivo experiments indicate that muscle contractions in the
animal are essential for synthesis of benzoyl cyanide.
We propose the following molecular defense system for this invasive animal (Fig.4e): when caught by a large
predator, the millipede severely contracts its body muscle, which results in extremely high blood pressure. Aer
endogenously rupturing the membranes of the storage and reaction chambers, the ChuaMOX in the blood
immediately ows into these chambers due to the high blood pressure. e suppressed enzyme is activated by
the shi of pH from 7 to 4.6 (see Supplementary Fig. S6), which starts the synthesis of benzoyl cyanide from
(R)-mandelonitrile. Finally, the benzoyl cyanide is released from the sacriced body, acting as a toxic exudate.
ese defensive chemicals provide protection to both the animal under attack and its swarm. In some cases,
the continuous release of the defensive chemicals from the prey contributes to the protection of its undamaged
swarm from the large predator. Among eusocial insects, the carpenter ant, Camponotus cylindricus complex, also
shows, endogenously rupturing its mandibular glands by body muscle contraction to exude defensive secretions
Figure 2. Phylogenetic analysis of ChuaMOX. ChuaMOX is shown in bold. e accession numbers of glucose
dehydrogenases and alcohol dehydrogenases appear in parentheses. Bootstrap values were determined from
1,000 replications. A bar indicates a 5% divergence.
www.nature.com/scientificreports/
6
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
of polyacetate-derived aromatics, aliphatic hydrocarbons, and alcohols from the mandibular gland2. Soldiers of
the termite, Globitermes sulphureus, contract their body muscle, rupture the frontal gland membrane, and exude
the defensive secretions3. e invasive millipede, C. hualienensis, is a larger arthropod than ants and termites. Its
size (3.5 cm) makes it an appropriate pray for wild birds. Although it is not a eusocial animal with sophisticated
castes, it also shows rupturing the defensive sacs, which consequently plays a role in protecting its swarm. In the
eld observation during millipede collection in 2013–2015, dusky thrushes approached the gutter lled with
the millipedes. However, the wild birds showed just hopping several times toward the gutter, never attacked the
swarm, and ew away all the time. Presumably they already learned that the millipedes were not eatable because
of its defense system. us, the habitat of this millipede may be expanding in Japan yearly.
Insects and millipedes diverged into Chelicerata and Myriapoda 666 million years ago19. e basal lineages of
ants and termites diverged approximately 105–110 million years ago and 130 million years ago, respectively20, and
each developed eusocial forms. Self-destructive altruistic defenses have evolved independently in each eusocial
form in response to various enemies4. Although this defensive strategy has been considered as a system unique to
social animal, the invasive primitive millipede, which has not evolved eusociality, is also equipped with, rupturing
the defensive sacs to protect its swarm. Further study on the defense systems in primitive arthropods will pave the
way to delineate the establishment of altruistic defenses in the animal kingdom.
Materials and Methods
Millipede. Invasive millipedes of C. hualienensis [Polydesmida: Paradoxomatidae] were collected in Japanese
cedar forests in southern Japan in 2013–2015 and shipped to our lab. e colony was maintained by supplying it
with sliced sweet potatoes and water at 25 °C and 70–90% relative humidity. Alternatively, sampled animals were
frozen with dry ice and stored at 80 °C until use.
Figure 3. Localization of ChuaMOX and its substrate. (a) Expression of ChuaMOX. RT-PCR detects
the gene expression in the paraterga, and actin expression is used as an internal control. (b) Localization
of ChuaMOX. e enzyme activity was mainly detected in the blood by zymography. 1, Antenna. 2, Leg. 3,
Head. 4, Integument. 5, Paraterga. 6, Fat body. 7, Gut. 8, Blood. (c) Localization of ChuaMOX substrate. (R)-
Mandelonitrile is detected in the extract from the paraterga. Blue, Antenna. Light blue, Leg. Green, Head and
tail. Purple, Integument. Red, Paraterga. Orange, Gut and fat body. Each extracted sample was analyzed using
an HPLC equipped with a chiral column, and the inset shows a magnied view of the chromatogram for a
retention time between 10 min and 15 min. Arrows indicate the peaks for benzaldehyde and (R)-mandelonitrile,
respectively. Based on the calibration curves, the estimated amounts of benzaldehyde and (R)-mandelonitrile
are 121 nmol and 93 nmol, respectively. (d) Whole body extract. e production of benzaldehyde, benzoyl
cyanide, and benzoic acid detected by GC/MS analysis is indicated by arrows. (e) Blood extract. Cyanohydrins
were not detected as substrates for ChuaMOX by GC/MS analysis.
www.nature.com/scientificreports/
7
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
Measurement of enzymatic activity. e activity of ChuaMOX was measured by monitoring the rate
of hydrogen peroxide formation using 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt)
(ABTS) (Dojindo, Kumamoto, Japan)21. e following reaction solution (1 ml) was prepared: 100 mM citrate
buer, pH 5, 0.1 mM ABTS, 5 mM racemic mandelonitrile (Sigma-Aldrich, St. Louis, MO, USA), and 150 mU
horseradish peroxidase (Wako Pure Chemical Industries, Osaka, Japan). Following 5 min of preincubation, the
enzyme solution was added to the reaction solution and mixed gently. e formation of hydrogen peroxide was
measured by monitoring its absorbance at 405 nm and 25 °C for 1 min using a spectrophotometer (Evolution 201
UV-visible Spectrophotometer, ermo Fisher Scientic, Waltham, MA, USA). Each point represents the mean
value of three independent experiments, and one unit of activity is dened as the amount of the enzyme needed
to catalyze the production of 1 μ mol of hydrogen peroxide for 1 min.
Figure 4. Synthesis of benzoyl cyanide as a defensive secretion in vivo. (a) Extract from a whole body.
(b) Extract from a whole body aer shaking. e arrows indicate peaks for benzaldehyde, benzoyl cyanide, and
(R)-mandelonitrile, respectively. (c) Extract from a whole body aer anesthetizing. e anesthetized animal is
not able to release the defensive compounds including benzoyl cyanide. (d) Extraction from paraterga collected
from the weakly anesthetized animal. e defensive sac in the paraterga was presumably ruptured by roughly
snatching collection and residual blood and mandelonitrile were mixed. e isolated tissues synthesized
benzoyl cyanide as non-anesthetized animals in (a,b). (e) Proposed defensive reaction of the millipede
through ChuaMOX. A millipede caught by a predator strongly contracts its body muscles, and the behavior
endogenously ruptures the membranes of the storage and reaction chambers. Drone blood enzyme, ChuaMOX,
ows into the chambers from the hemocoel and is activated by the shi of pH from 7 to 4.6, which starts the
synthesis of benzoyl cyanide from (R)-mandelonitrile.
www.nature.com/scientificreports/
8
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
Purication of ChuaMOX from the invasive millipede. Frozen millipedes were thawed at room tem-
perature, and the gut was removed using ne forceps under a microscope. e bodies without guts were homog-
enized in 10 mM Tris-HCl, pH 8, and centrifuged to remove debris.
All purification steps were carried out at 4 °C. The crude extract was loaded onto DEAE Sepharose FF
(GE Healthcare, Little Chalfont, UK) and eluted with 10 mM Tris-HCl, pH 8, containing 50 mM sodium chloride.
e diluted positive fraction was loaded onto Q Sepharose FF (GE Healthcare) and eluted with 10 mM Tris-HCl,
pH 8, containing 25 mM sodium chloride. e concentrated sample was loaded onto Superdex 200 10/300 GL
(GE Healthcare) and eluted with 20 mM Tris-HCl, pH 8, containing 150 mM sodium chloride at a ow rate of
0.5 ml/min. e puried enzyme is stable in 20 mM Tris-HCl, pH 8 at 4 °C for 6 months.
e protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA,
USA) with bovine serum albumin as the standard protein. To estimate the molecular mass of the mandelonitrile
oxidase, 10% SDS-PAGE and gel ltration were used.
UV and visible spectral analysis. The absorption spectrum of the ChuaMOX (1 mg/ml in 10 mM
Tris-HCl, pH 8) was recorded using a spectrophotometer (Evolution 201 UV-visible Spectrophotometer, ermo
Fisher Scientic).
Thin layer chromatography analysis. in layer chromatography (TLC) analysis was followed using the
methodology of the previous publication22.
Detection of sugar molecules in ChuaMOX. Aer separation on 10% SDS-PAGE, the sugar molecules
in ChuaMOX were detected by the periodic acid-Schi (PAS) method using a Pierce Glycoprotein Staining Kit
(ermo Fisher Scientic).
Characterization of ChuaMOX. e chemicals (see Supplementary Table S5) were purchased from vari-
ous suppliers (Sigma-Aldrich, Dojindo, Alfa Aesar, and Tokyo Chemical Industries) or synthesized as described
below and used as test substrates, inhibitors, and metal salts. Nitriles were prepared from their corresponding
aldehydes by way of bisulte adducts according to Young et al.23.
To determine optimum pH and pH stability, 100 mM of citrate buer of pH 3.0–6.0, phosphate buer of pH
6.0–8.0, Tris-HCl of pH 8.0–9.0, and glycine-sodium hydroxide of pH 9.0–10.0 were used. Enzyme activity was
measured for a range of 10 °C to 70 °C to determine the optimum temperature and temperature stability. A 1-hour
preincubation without substrate was used in the assays for pH stability and temperature stability and the other
procedures were carried out as described above.
Synthesis of benzoyl cyanide using the puried ChuaMOX. Benzoyl cyanide, (R)- and (S)-mandelonitrile,
and benzaldehyde were used in known amounts of authentic chemicals to estimate retention time and prepara-
tion of calibration curves (see Supplementary Fig. S2). Because a millipede contains 1 μ mol of (R)-mandelonitrile
and 0.1 U of ChuaMOX in 100 μ l of blood15 according to the calibration curve and the purication table, the same
amounts of mandelonitrile and ChuaMOX were mixed in 100 μ l of 100 mM of a pH 5 citrate buer and incubated
at 25 °C for 1 min. e product was analyzed using an HPLC equipped with a chiral column (CHIRALCEL OJ-H
column: particle size, 5 μ m; 4.6 mm i.d. × 250 mm; Daicel, Osaka, Japan)8.
Measurement of oxygen consumption. Oxygen consumption in the enzyme reaction (nmol/ml/min)
was monitored using a S1 Clark type polarographic electrode24 connected with an Oxygraph Plus oxygen elec-
trode system (Hansatech Instruments, Norfolk, UK).
Zymography. A millipede was completely anesthetized on ice, and blood was collected from small
wounds. The paraterga were collected by fine forceps under a microscope. After immersing the body in a
phosphate-buered saline (PBS), other tissues were collected. Each tissue was homogenized in 10 mM Tris-HCl,
pH 8. Protein extract equivalent to 1 animal of antenna, leg, head, integument, paraterga, and gut; 0.25 animal
equivalent of fat body; and 0.1 animal equivalent of blood were loaded and separated on 10% native PAGE.
Zymography was performed as follows: the gel was developed in 50 mM citrate buer, pH 5 containing 0.6 mM
2-(4-indophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolim chloride (INT) (Dojindo Laboratories), 0.33 mM
1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) (Dojindo Laboratories), and 10 mM racemic
mandelonitrile (Sigma-Aldrich).
Detection of benzoyl cyanide as a defensive secretion from the millipede and the paraterga.
Each millipede was treated with one of the following treatment: no treatment (control), shaking for 2 min, or anes-
thesia using diethyl ether. Aer each treatment, each animal was soaked in organic solvent (n-hexane:2-propanol
with a volume ration of 85:15) for 3 min, and the extract was analyzed using an HPLC equipped with a chiral col-
umn8. On the other hand, the paraterga was collected from the weakly anesthetized animals by roughly snatching
using a ne forceps to rupture the defensive sac and mix mandelonitrile and blood containing ChuaMOX.
Gas chromatography-mass spectrometric analysis. A millipede or 10 μ l of blood were transferred
into a glass vial and immersed in n-hexane (5 ml) for 3 min at room temperature. e n-hexane extract (4 μ l
portion, each) was analyzed using a gas chromatography-mass spectrometer (GC/MS) (7890A GC System cou-
pled with a 5975C inert XL EI/CI MSD with a Triple-Axis Detector operated at 70 eV; Agilent Technologies,
Santa Clara, CA, USA) equipped with an HP-5 ms capillary column (0.25 mm i.d. × 30 m, 0.25 μ m lm thickness;
Agilent Technologies) according to the previous publication13.
www.nature.com/scientificreports/
9
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
Protein sequencing. After the separation of the purified ChuaMOX on 10% SDS-PAGE, the protein
was visualized by Coomassie Brilliant Blue staining. e protein band was excised and treated with trypsin
(sequencing-grade modied trypsin; Promega, Madison, WI, USA) following the previously reported meth-
ods25,26. e digested peptides were separated using a nanoUPLC equipped with a trap column (nanoACQUITY
UPLC Symmetry C18 Trap Column, 100 Å, 5 μ m, 180 μ m x 20 mm, Waters, Milford, MA, USA) and a reverse
phase capillary column (ACUITY UPLC Peptide CSH C18 nanoACQUITY Column 10 K psi, 130 Å, 1.7 μ m ,
75 μ m × 200 mm, Waters). e molecular mass was determined with an electrospray ionization/quadrupole
time-of-ight mass spectrometer (nanoESI-SYNAPT G2-Si mass spectrometry; Waters). e MS/MS spectra
were processed using the Biolynx soware suite (Waters).
Transcriptome analysis. Total RNA was extracted from the animals without guts using a TRIzol Reagent
(ermo Fisher Scientic). Aer treatment with DNase I, the total RNA was re-puried using the RNeasy Mini
Kit (Qiagen, Valencia, CA, USA), and the RNA samples were frozen with dry ice and shipped to Hokkaido System
Science (Sapporo, Japan). Aer quality verication of the total RNA using an Agilent 2100 Bioanalyzer (Agilent
Technologies), the cDNA library was constructed. To reduce the highly abundant transcripts before pyrose-
quencing, the library was normalized using the TRIMMER DIRECT cDNA normalization kit (Evrogen, Moscow,
Russia) and pyrosequencing, including library construction, was performed at Hokkaido System Science. In brief,
the raw reads sequenced using a GS FLX + system (Roche 454 Company, Branford, CT, USA) were cleaned by
removing the adaptor sequence and unknown or low quality bases. De novo transcriptome assembly was per-
formed using a GS De Novo Assembler v2.8 (Roche Applied Bioscience, Penzberg, Germany) with its default
settings, and the assembled sequences were deposited in our local BLAST server. e C. hualienensis sequencing
and assembly were summarized in Supplementary Table S6. e raw reads of sequence data was deposited in the
DDBJ Sequence Read Archive (BioProject ID, PRJDB3791).
cDNA cloning. A partial cDNA sequence encoding 5 amino acid sequences determined using a quadruple
time-of-ight mass spectrometer was obtained from an in-house EST data base. Total RNA was isolated from the
paraterga of C. hualienensis using a TRIzol Reagent (ermo Fisher Scientic), and cDNA was synthesized using
a SMART RACE cDNA Amplication Kit (Takara Bio, Kusatsu, Japan), a 5 -Full RACE Core Set (Takara Bio), and
a GeneRacer Kit (ermo Fisher Scientic). PCR was carried out using KOD plus neo (Toyobo, Osaka, Japan)
and the gene-specic primers (see Supplementary Table S7), and the amplicons were sequenced using a DNA
sequencer (3500 Genetic analyzer, ermo Fisher Scientic). e full-length cDNA sequence was determined
using 21 independent clones to avoid PCR-derived sequence errors, and the DNA and amino acid sequences of
ChuaMOX were analyzed using GENETYX ver. 11 and ATGC (Genetyx, Tokyo, Japan), PeptideMass27, NetNGlyc
1.0 Server28, Cofactory29, and MEGA630.
RT-PCR. Each experimental tissue was collected as described above and immediately homogenized in a TRIzol
Reagent (ermo Fisher Scientic) aer the addition of glycogen (ermo Fisher Scientic). cDNA was synthe-
sized using a SMART RACE cDNA Amplication Kit (Takara Bio) and SuperScript II (ermo Fisher Scientic) as
a reverse transcriptase. e gene-specic primers, ChuaMOX-1 and ChuaMOX-2 (see Supplementary Table S7),
were used for RT-PCR. e expression of actin was detected as an internal control31.
Measurement of the pH of blood and secretion from the ozopore. e pH of the solutions was
estimated using pH indicator papers (Macherey-Nagel, Düren, Germany) according to previous publications13,14.
References
1. Holldobler, B. & Wilson, E. O. the ANTS. (Belanap Press of Harvard University Press, 1990).
2. Jones, T. H. et al. e chemistry of exploding ants, Camponotus spp. ( cylindricus complex). J. Chem. Ecol. 30, 1479–1491 (2004).
3. Bordereau, C., obert, A., Tuyen, V. V. & Peppuy, A. Suicidal defensive behaviour by frontal gland dehiscence in Globitermes
sulphureus Haviland soldiers (Isoptera). Insectes Soc. 44, 289–296 (1997).
4. Shorter, J. . & ueppell, O. A review on self-destructive defense behaviors in social insects. Insectes Soc. 59, 1–10 (2012).
5. Blum, M. S. Chemical defenses of arthropods. (Academic Press, 1981).
6. Eisner, T. & Meinwald, J. Defensive secretions of arthropods. Science 153, 1341–1350 (1966).
7. Asano, Y. et al. Screening for new hydroxynitrilases from plants. Biosci. Biotechnol. Biochem. 69, 2349–2357 (2005).
8. Dadashipour, M., Ishida, Y., Yamamoto, . & Asano, Y. Discovery and molecular and biocatalytic properties of hydroxynitrile lyase
from an invasive millipede, Chamberlinius hualienensis. Proc. Natl. Acad. Sci. USA 112, 10605–10610 (2015).
9. Asano, Y. Overview of screening for new microbial catalysts and their uses in organic synthesis: Selection and optimization of
biocatalysts. J. Biotechnol. 94, 65–72 (2002).
10. Yamaguchi, T., Yamamoto, . & Asano, Y. Identication and characterization of CYP79D16 and CYP71AN24 catalyzing the rst and
second steps in phenylalanine-derived cyanogenic glycoside biosynthesis in the Japanese apricot, Prunus mume Sieb. et Zucc. Plant
Mol. Biol. 86, 215–223 (2014).
11. Chen, C.-C., Golovatch, S. I., Chang, H.-W. & Chen, S.-H. evision of the Taiwanese millipede genus Chamberlinius Wang, 1956,
with descriptions of two new species and a reclassication of the tribe Chamberlinini (Diplopoda, Polydesmida, Paradoxosomatidae,
Paradoxosomatinae). Zooeys 98, 1–27 (2011).
12. Noguchi, S., Mori, N., Higa, Y. & uwahara, Y. Identication of mandelonitrile as a major secretory compound from Chamberlinius
hualienensis Wang (Polydesmida: Paradoxosomatidae). Jpn. J. Environ. Entomol. Zool. 8, 208–214 (1997).
13. uwahara, Y., Shimizu, N. & Tanabe, T. elease of hydrogen cyanide via a post-secretion Schotten-Baumann reaction in defensive
uids of polydesmoid millipedes. J. Chem. Ecol. 37, 232–238 (2011).
14. Duey, S. S. et al. Benzoyl cyanide and mandelonitrile benzoate in the defensive secretions of millipedes. J. Chem. Ecol. 3, 101–113
(1977).
15. Nation, J. L. Insect Physiology and Biochemistry. (CC Press, 2002).
16. leiger, G. & Eisenberg, D. GXXXG and GXXXA motifs stabilize FAD and NAD(P)-binding ossmann folds through Ca–H· · ·O
hydrogen bonds and van der waals interactions. J. Mol. Biol. 323, 69–76 (2002).
17. Bonasio, . et al. Genomic comparison of the ants Camponotus oridanus and Harpegnathos saltator. Science 329, 1068–1071
(2010).
www.nature.com/scientificreports/
10
Scientific RepoRts | 6:26998 | DOI: 10.1038/srep26998
18. Eisner, T., Eisner, M., Attygalle, A. B., Deyrup, M. & Meinwald, J. endering the inedible edible: Circumvention of a millipede’s
chemical defense by a predaceous beetle larva (Phengodidae). Proc. Natl. Acad. Sci. USA 95, 1108–1113 (1998).
19. Pisani, D., Poling, L. L., Lyons-Weiler, M. & Hedges, S. B. e colonization of land by animals: molecular phylogeny and divergence
times among arthropods. BMC Biol. 2, 1 (2004).
20. orne, B. L. & Traniello, J. F. A. Comparative social biology of basal taxa of ants and termites. Ann. ev. Entomol. 48, 283–306
(2003).
21. eesey, J. Biochemica Information: A revised biochemical research source. 1st edn, (Boehringer Mannheim Biochemicals, 1987).
22. Isobe, . & Nagasawa, S. Characterization of Nα-benzyloxycarbonyl- L-lysine oxidizing enzyme from hodococcus sp. AIU Z-35-1.
J. Biosci. Bioeng. 104, 218–223 (2007).
23. Young, S. D., Buse, C. T. & Heathcoc, C. H. 2-Methyl-2-(trimethylsiloxy)pentan-3-one [3-pentanone, 2-methyl-2-[(trimethylsilyl)
oxy]-]. Org. Synth. 63, 79 (1985).
24. Clar, L. C. Monitor and control of blood and tissue O2 tensions. Trans. Am. Soc. Artif. Intern. Organs 2, 41–48 (1956).
25. Shevcheno, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide
gels. Anal. Chem. 68, 850–858 (1996).
26. Wilm, M. & Mann, M. Analytical properties of the nanoelectrospray ion source. Anal. Chem. 68, 1–8 (1996).
27. Gasteiger, E. et al. In e Proteomics Protocols Handboo (ed John M. Waler) 571–607 (Humana Press, 2005).
28. Gupta, . & Bruna, S. In Pacic Symposium on Biocomputing Vo l. 7 310–322 (2002).
29. Geertz-Hansen, H. M., Blom, N., Feist, A. M., Bruna, S. & Petersen, T. N. Cofactory: Sequence-based prediction of cofactor
specicity of ossmann folds. Proteins: Struct., Funct., Bioinf. 82, 1819–1828 (2014).
30. Tamura, ., Stecher, G., Peterson, D., Filipsi, A. & umar, S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol.
Biol. Evol. 30, 2725–2729 (2013).
31. Ishida, Y. & Leal, W. S. Cloning of putative odorant-degrading enzyme and integumental esterase cDNAs from the wild silmoth,
Antheraea polyphemus. Insect Biochem. Mol. Biol. 32, 1775–1780 (2002).
Acknowledgements
is work was supported by the Exploratory Research for Advanced Technology (ERATO) program of the Japan
Science and Technology Agency (JST). We would like to extend great thanks to Dr. K. Isobe for suggesting the
experiments and for providing valuable comments during the preparation of our manuscript. e ChuaMOX
cDNA nucleotide sequence data was deposited in the DNA Data Bank of Japan (DDBJ) (accession number,
LC036560), and the transcriptome sequence data were deposited in the DDBJ Sequence Read Archive (BioProject
ID, PRJDB3791).
Author Contributions
Y. Ishida, Y.K., M.D., T.Y., M.M., Y. Ichiki and Y.A. collected millipedes. Y. Ishida contributed the experimental
design and performed isolation, characterization, and localization of ChuaMOX and data analysis. Y.K. performed
the GC/MS analysis and data analysis. M.D. performed the measurement and localization of mandelonitrile in
the millipede and data analysis. A.I. performed the protein sequencing and data analysis. T.Y. performed the
transcriptome analysis and data analysis. M.M. performed the synthesis of cyanohydrins. Y. Ichiki performed the
preliminary experiments. Y.A. contributed the experimental design and data analysis.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Ishida, Y. et al. A sacricial millipede altruistically protects its swarm using a drone
blood enzyme, mandelonitrile oxidase. Sci. Rep. 6, 26998; doi: 10.1038/srep26998 (2016).
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
... known to belong to the AA3_2 subfamily, and (R)-mandelonitrile oxidase (EC 1.1.3.49), which is annotated as a member of the same subfamily in the holobiont of T. aoutii. The latter, characterized in the polydesmid millipede Chamberlinius hualienensis, is involved in the production of the defense chemical benzoyl cyanide (Ishida et al., 2016). The superoxide dismutase (SOD) and tyrosinase (hemocyanin), both Heat maps of CAZy families, expressed in the host and prevailing microbiota group (Bacteria), and their distribution among the gut segments in the holobiont. ...
Article
Full-text available
As important decomposers of soil organic matter, millipedes contribute to lignocellulose decomposition and nutrient cycling. The degradation of lignocellulose requires the action of several carbohydrate-active enzymes (CAZymes) and, in most invertebrates, depends on the activity of mutualistic gut microorganisms. To address the question of the importance of the microbiota and endogenous (host) enzymes in digestive processes in millipedes, we analyzed metatranscriptomic data from the tropical millipede Telodeinopus aoutii at the holobiont level. Functional annotation included identification of expressed CAZymes (CAZy families and EC terms) in the host and its intestinal microbiota, foregut, midgut, and hindgut, compared to non-intestinal tissues. Most of the 175 CAZy families were expressed exclusively in the gut microbiota and more than 50% of these microbial families were expressed exclusively in the hindgut. The greatest diversity of expressed endogenous CAZymes from all gut sections was found in the midgut (77 families). Bacteria were the major microbial producers of CAZymes, Proteobacteria dominating in the midgut and Bacteriodetes with Firmicutes in the hindgut. The contribution of the eukaryotic microbiota to CAZymes production was negligible. Functional classification of expressed CAZy families confirmed a broad functional spectrum of CAZymes potentially expressed in the holobiont. Degradation of lignocellulose in the digestive tract of the millipede T. aoutii depends largely on bacterial enzymes expressed in the hindgut. Endogenous cellulases were not detected, except for the potentially cellulolytic family AA15, but an expression of cellulolytic enzymes of this family was not confirmed at the EC-number level. The midgut had the greatest diversity of expressed endogenous CAZymes, mainly amylases, indicating the importance of digesting α-glucosidases for the millipede. In contrast, bacterial lignocellulolytic enzymes are sparsely expressed here. The hindgut was the hotspot of microbial degradation of cellulose and hemicellulases. The gain of the millipede from the microbial lignocellulose degradation in the gut, and consequently the mutualistic status of the relationship between the millipede and its cellulolytic gut bacteria, depends on the ability of the millipede to take up microbial metabolites as nutrients through the hindgut wall. Enzymes expressed in the intestine can degrade all components of lignocellulose except lignin. Assuming that soil microbiota is partially degraded lignin in the millipede diet, T. aoutii can be considered a decomposer of soil organic matter relying primarily on its gut bacteria. The deposition of millipede fecal pellets containing an organic matter modified by the hindgut bacterial community could be of ecological significance.
... 10 Another enzyme is mandelonitrile oxidase (ChuaMOX), which acts on (R)-mandelonitrile to produce benzoyl cyanide and hydrogen peroxide. 21 Based on our knowledge, polydesmid millipedes can be a target for further discovery and identification of biocatalysts and their respective functions. ...
Article
Full-text available
Hydroxynitrile lyase (HNL) catalyzes the reversible synthesis and degradation of cyanohydrins, which are important synthetic intermediates for fine chemical and pharmaceutical industries. Here, we report the discovery of HNL from Parafontaria laminata (PlamHNL) millipedes, purification of the HNL to homogeneity, expression of the gene for the enzyme in heterologous expression hosts, and increase in the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile by protein engineering. The recombinant PlamHNL expressed in Pichia pastoris is glycosylated and has a higher thermostability and pH stability than the nonglycosylated HNL expressed in Escherichia coli. PlamHNL showed a unique wide substrate specificity among other millipede HNLs acting on various cyanohydrins, including 2-chloromandelonitrile, a key intermediate for the antithrombotic agent clopidogrel. We solved the X-ray crystal structure of the PlamHNL and found that the catalytic residues were almost identical to those of HNL from Chamberlinius hualienensis, although the forming binding cavity was different. In order to improve the catalytic activity and stereoselectivity, a computational structure-guided directed evolution approach was performed by an enzyme−substrate docking simulation at all of the residues that were exposed on the surface of the active site. The PlamHNL-N85Y mutant showed higher conversion (91% conversion with 98.2% ee of the product) than the wild type (76% conversion with 90% ee of the product) at pH 3.5 and 25°C for 30 min of incubation. This study shows the diversity of millipede HNLs and reveals the molecular basis for improvement of the activity and stereoselectivity of the wild-type HNL to increase the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile.
... We established the molecular mechanisms of the synthesis of defense allomones and shown that adult millipedes produce a mixture 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 mandelonitrile 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]. ...
Article
Full-text available
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 (7,420 U/mg) 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 Å 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.
... We established the molecular mechanisms of the synthesis of defense allomones, and shown that adult millipedes produce a mixture 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 mandelonitrile oxidase [31]. Benzoyl cyanide further accepts one more molecule of (R)-MAN to form an ester (R)-MAN benzoate by a non-enzymatic Schotten-Bauman reaction releasing one molecule of cyanide [32]. ...
Article
Full-text available
We report the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL‐Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL‐Np) isolated and purified from passion fruit leaves. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99% identical with one amino acid difference. Purple passion fruit PeHNL‐Np has Ala residue at position 107 and is non‐glycosylated at Asn105. As it was confirmed that natural and glycosylated Pe HNL‐Ny showed superior thermostability, pH stability, and organic tolerance than the PeHNL‐Np, we speculated that protein engineering around Asn105 located at the C‐terminal region of PeHNL‐Ny might contribute to stabilization of PeHNL. Therefore, we focused on the improvement of stability of the non‐glycosylated PeHNL by truncating its C‐terminal region. The C‐terminal truncated PeHNLΔ 107 was obtained by truncating 15 amino acids from its C‐terminal and was expressed in Escherichia coli. PeHNLΔ 107 expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to the wild‐type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high flexibility region at the C‐terminal of PeHNL might be the possible reason for improving the stability of PeHNL.
... AJ302014). To obtain PDIs from C. hualienensis, a homology search against PpPDI was performed using BLAST against the RNA sequence data of C. hualienensis [11]. As a result, two candidate PDI genes, ChuaPDI1 and ChuaPDI2, were isolated. ...
Article
A hydroxynitrile lyase (HNL) from the millipede Chamberlinius hualienensis has high potential for industrial use in the synthesis of cyanohydrins. However, obtaining sufficient amounts of millipedes is difficult, and the production of the Chamberlinius hualienensis HNL (ChuaHNL) in E. coli has not been very successful. Therefore, we investigated the conditions required for high-yield heterologous production of this enzyme using Pichia pastoris. When we employed P. pastoris to express His-ChuaHNL, the yield was very low (22.6 ± 3.8 U/L culture). Hence, we investigated the effects of ChuaHNL codon optimization and the co-production of two protein disulfide isomerases (PDIs) [from P. pastoris (PpPDI) and C. hualienensis (ChuaPDI1, ChuaPDI2)] on His-ChuaHNL production. The productivity of His-ChuaHNL was increased approximately 140 times per unit culture to 3170 ± 144.7 U/L by the co-expression of codon-optimized ChuaHNL and PpPDI. Moreover, we revealed that the N-glycosylation on ChuaHNL had a large effect on the stability, enzyme secretion, and catalytic properties of ChuaHNL in P. pastoris. This study demonstrates an economical and efficient approach for the production of HNL, and the data show that glycosylation has a large effect on the enzyme properties and the P. pastoris expression system.
... We do not have exact information about the most substantially downregulated enzyme in our study, glucose dehydrogenase [FAD, quinone]-like (EC:1.1.3.49); however, according to CCD, it belongs to the choline dehydrogenase and glucose-methanol-choline oxidoreductase (GMCOX) family and thus may participate in choline metabolism [89] and/or conversion of (R)-mandelonitrile to benzoyl cyanide [90]. In the context of the present study, it appears important that the GMCOX C- terminal domain is involved in steroid binding [91,92], and thus the reduced abundance of this protein is potentially connected to impaired sterol synthesis. ...
Article
Determining the side effects of pesticides on pollinators is an important topic due to the increasing loss of pollinators. We aimed to determine the effects of chronic sublethal exposure of the neonicotinoid pesticide imidacloprid on the bumblebee Bombus terrestris under laboratory conditions. The analytical standard of imidacloprid in sugar solution was used for the treatment. Verification of pesticides using UHPLC-QqQ-MS/MS in the experimental bumblebees showed the presence of only two compounds, imidacloprid and imidacloprid-olefin, which were found in quantities of 0.57±0.22 and 1.95±0.43 ng/g, respectively. Thus, the level of the dangerous metabolite imidacloprid-olefin was 3.4-fold higher than that of imidacloprid. Label-free nanoLC-MS/MS quantitative proteomics of bumblebee heads enabled quantitative comparison of 2,883 proteins, and 206 proteins were significantly influenced by the imidacloprid treatment. The next analysis revealed that the highly downregulated markers are members of the terpenoid backbone biosynthesis pathway (KEGG: bter00900) and that imidacloprid treatment suppressed the entire mevalonate pathway, fatty acid synthesis and associated markers. The proteomics results indicate that the consequences of imidacloprid treatment are complex, and the marker changes are associated with metabolic and neurological diseases and olfaction disruption. This study provides important markers and can help to explain the widely held assumptions from biological observations. Significance: The major finding is that all markers of the mevalonate pathway were substantially downregulated due to the chronic imidacloprid exposure. The disbalance of mevalonate pathway has many important consequences. We suggest the mechanism associated with the novel toxicogenic effect of imidacloprid. The results are helpful to explain that imidacloprid impairs the cognitive functions and possesses the delayed and time cumulative effect.
... Changes in these markers indicate a violation of communication in the colony and can explain the previously reported adverse effects of imidacloprid on olfactory learning [17,18]. [89] and/or conversion of (R)-mandelonitrile to benzoyl cyanide [90]. In the context of the present study, it appears important that the GMCOX Cterminal domain is involved in steroid binding [91,92], and thus the reduced abundance of this protein is potentially connected to impaired sterol synthesis. ...
Article
Determining the side effects of pesticides on pollinators is an important topic due to the increasing loss of pollinators. We aimed to determine the effects of chronic sublethal exposure of the neonicotinoid pesticide imidacloprid on the bumblebee Bombus terrestris under laboratory conditions. The analytical standard of imidacloprid in sugar solution was used for the treatment. Verification of pesticides using UHPLC-QqQ-MS/MS in the experimental bumblebees showed the presence of only two compounds, imidacloprid and imidacloprid-olefin, which were found in quantities of 0.57±0.22 and 1.95±0.43 ng/g, respectively. Thus, the level of the dangerous metabolite imidacloprid-olefin was 3.4-fold higher than that of imidacloprid. Label-free nanoLC-MS/MS quantitative proteomics of bumblebee heads enabled quantitative comparison of 2,883 proteins, and 206 proteins were significantly influenced by the imidacloprid treatment. The next analysis revealed that the highly downregulated markers are members of the terpenoid backbone biosynthesis pathway (KEGG: bter00900) and that imidacloprid treatment suppressed the entire mevalonate pathway, fatty acid synthesis and associated markers. The proteomics results indicate that the consequences of imidacloprid treatment are complex, and the marker changes are associated with metabolic and neurological diseases and olfaction disruption. This study provides important markers and can help to explain the widely held assumptions from biological observations. Significance: The major finding is that all markers of the mevalonate pathway were substantially downregulated due to the chronic imidacloprid exposure. The disbalance of mevalonate pathway has many important consequences. We suggest the mechanism associated with the novel toxicogenic effect of imidacloprid. The results are helpful to explain that imidacloprid impairs the cognitive functions and possesses the delayed and time cumulative effect.
... A total of 20 peaks (11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,27,28,29,30, and 31, 45.4%) were newly detected in the present species and represented the first discovery of the hereafter named "waxy compounds" among Polydesmida. Their structures were later elucidated as 1-methoxyalkanes by (1) column chromatography (SiO 2 ) behavior, (2) NMR analysis, and (3) GC-mass spectra (the presence of M + or M + -32, and m/z 45), using 1-methoxyalkanes prepared from palm oil as an authentic compound. ...
Article
Full-text available
Mixtures of saturated and unsaturated 1-methoxyalkanes (alkyl methyl ethers, representing more than 45.4% of the millipede hexane extracts) were newly identified from the Thai polydesmid millipede, Orthomorpha communis, in addition to well-known polydesmid defense allomones (benzaldehyde, benzoyl cyanide, benzoic acid, mandelonitrile, and mandelonitrile benzoate) and phenolics (phenol, o- and p-cresol, 2-methoxyphenol, 2-methoxy-5-methylphenol and 3-methoxy-4-methylphenol). The major compound was 1-methoxy-n-hexadecane (32.9%), and the mixture might function as "raincoat compounds" for the species to keep off water penetration and also to prevent desiccation.
Article
Full-text available
The Myriapoda, composed of millipedes and centipedes, is a fascinating but poorly understood branch of life, including species with a highly unusual body plan and a range of unique adaptations to their environment. Here, we sequenced and assembled 2 chromosomal-level genomes of the millipedes Helicorthomorpha holstii (assembly size = 182 Mb; shortest scaffold/contig length needed to cover 50% of the genome [N50] = 18.11 Mb mainly on 8 pseudomolecules) and Trigoniulus corallinus (assembly size = 449 Mb, N50 = 26.78 Mb mainly on 17 pseudomolecules). Unique genomic features, patterns of gene regulation, and defence systems in millipedes, not observed in other arthropods, are revealed. Both repeat content and intron size are major contributors to the observed differences in millipede genome size. Tight Hox and the first loose ecdysozoan ParaHox homeobox clusters are identified, and a myriapod-specific genomic rearrangement including Hox3 is also observed. The Argonaute (AGO) proteins for loading small RNAs are duplicated in both millipedes, but unlike in insects, an AGO duplicate has become a pseudogene. Evidence of post-transcriptional modification in small RNAs-including species-specific microRNA arm switching-providing differential gene regulation is also obtained. Millipedes possesses a unique ozadene defensive gland unlike the venomous forcipules found in centipedes. We identify sets of genes associated with the ozadene that play roles in chemical defence as well as antimicrobial activity. Macro-synteny analyses revealed highly conserved genomic blocks between the 2 millipedes and deuterostomes. Collectively, our analyses of millipede genomes reveal that a series of unique adaptations have occurred in this major lineage of arthropod diversity. The 2 high-quality millipede genomes provided here shed new light on the conserved and lineage-specific features of millipedes and centipedes. These findings demonstrate the importance of the consideration of both centipede and millipede genomes-and in particular the reconstruction of the myriapod ancestral situation-for future research to improve understanding of arthropod evolution, and animal evolutionary genomics more widely.
Article
Full-text available
Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.
Article
Full-text available
We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements our RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
Article
Full-text available
Colony defense is a necessary but dangerous task for social insects, and nest defensive behaviors often lead to a premature death of the actor. As an extreme form of colony defense, self-sacrificial behaviors have evolved by kin selection in various social insects. Most self-sacrificial defensive mechanisms occur in response to an acute threat to the colony, but some behaviors are preemptive actions that avert harm to the colony. Self-sacrifice has also been observed as a form of preemptive defense against parasites and pathogens where individuals will abandon their normal colony function and die in self-exile to reduce the risk of infecting nestmates. Here, we provide an overview of the self-destructive defense mechanisms that eusocial insects have evolved and discuss avenues for future research into this form of altruism.
Article
Japanese apricot, Prunus mume Sieb. et Zucc., belonging to the Rosaceae family, produces as defensive agents the cyanogenic glycosides prunasin and amygdalin, which are presumably derived from l-phenylalanine. In this study, we identified and characterized cytochrome P450s catalyzing the conversion of l-phenylalanine into mandelonitrile via phenylacetaldoxime. Full-length cDNAs encoding CYP79D16, CYP79A68, CYP71AN24, CYP71AP13, CYP71AU50, and CYP736A117 were cloned from P. mume ‘Nanko’ using publicly available P. mume RNA-sequencing data, followed by 5′- and 3′-RACE. CYP79D16 was expressed in seedlings, whereas CYP71AN24 was expressed in seedlings and leaves. Enzyme activity of these cytochrome P450s expressed in Saccharomyces cerevisiae was evaluated by liquid and gas chromatography–mass spectrometry. CYP79D16, but not CYP79A68, catalyzed the conversion of l-phenylalanine into phenylacetaldoxime. CYP79D16 showed no activity toward other amino acids. CYP71AN24, but not CYP71AP13, CYP71AU50, and CYP736A117, catalyzed the conversion of phenylacetaldoxime into mandelonitrile. CYP71AN24 also showed lower conversions of various aromatic aldoximes and nitriles. The K m value and turnover rate of CYP71AN24 for phenylacetaldoxime were 3.9 µM and 46.3 min−1, respectively. The K m value and turnover of CYP71AN24 may cause the efficient metabolism of phenylacetaldoxime, avoiding the release of the toxic intermediate to the cytosol. These results suggest that cyanogenic glycoside biosynthesis in P. mume is regulated in concert with catalysis by CYP79D16 in the parental and sequential reaction of CYP71AN24 in the seedling.
Article
Obtaining optimal cofactor balance to drive production is a challenge in metabolically engineered microbial production strains. To facilitate identification of heterologous enzymes with desirable altered cofactor requirements from native content, we have developed Cofactory, a method for prediction of enzyme cofactor specificity using only primary amino acid sequence information. The algorithm identifies potential cofactor binding Rossmann folds and predicts the specificity for the cofactors FAD(H2 ), NAD(H) and NADP(H). The Rossmann fold sequence search is carried out using hidden Markov models whereas artificial neural networks are used for specificity prediction. Training was carried out using experimental data from protein-cofactor structure complexes. The overall performance was benchmarked against an independent evaluation set obtaining Matthews correlation coefficients of 0.94, 0.79 and 0.65 for FAD(H2 ), NAD(H) and NADP(H), respectively. The Cofactory method is made publicly available at http://www.cbs.dtu.dk/services/Cofactory. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.