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

Abstract

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 selfdestructive 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.

A novel cytochrome P450, CYP3201B1, is involved in (R)‐mandelonitrile biosynthesis in a cyanogenic millipede

“…S1) [16]. The pH of the defensive secretion of C. hualienensis and other cyanogenic millipedes is maintained at approximately 4.0 [13,23,54]. In our observation, (R)-MAN was stable at pH 5.5 (Fig.…”

Rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity

“…For this effort, we used the following four insoluble enzymes: Ch MOX, At ADC, Dm GDH, and Dm ODC. Libraries of 12,000 clones of each of these four enzymes were obtained by random mutagenesis, and their enzyme activities were determined by the color development method 21 , 23 to obtain soluble forms with enzyme activity. Several mutants with activity were obtained from each of the four enzymes, and the following amino acid substitutions were identified: V455D (GTT → GAT) in Ch MOX, K441L (AAG→TTG) in At ADC, V174D (GTG→GAT) in Dm GDH, and K117L (AAG→TTG) in Dm ODC.…”

“…The previously constructed plasmids pET-22b- chmox and pUC-18- supdh , were used for expression of the Ch MOX gene ( chmox ) and the Su PDH gene ( supdh ), respectively 20 , 21 . The cDNA of Mp LUC was synthesized and amplified using PfuUltra II fusion HS DNA polymerase (Agilent Technologies, Santa Clara, CA, USA) and primers P1 and P2 (Table S1 ).…”

“…Ch MOX activity towards ( R )-mandelonitrile was assayed by the color development method 21 . Activities of At ADC towards l-arginine and Dm ODC towards l-ornithine were assayed by the color development method, coupled with putrescine oxidase from Micrococcus rubens IFO 3768 23 .…”

Hydroxynitrile lyases from cyanogenic millipedes: molecular cloning, heterologous expression, and whole-cell biocatalysis for the production of (R)-mandelonitrile

“…The N-terminal amino acid sequencing of purified NttHNL was performed at Nippi Inc. (Tokyo, Japan) using a Procise492HT protein sequencer (Applied Biosystems, Foster city, CA, USA). The internal amino acid sequence was determined as described previously 43 . Briefly, after separation of purified NttHNL by 12% SDS-PAGE, the protein was visualized by Coomassie Brilliant Blue staining, excised, and then treated with trypsin (sequencing-grade modified trypsin; Promega, Madison, WI, USA).…”