Some plants protect themselves with cyanide against voracious beetles, caterpillars or cattle, who want to nibble their delicious looking leaves. Everyone knows that cyanide is really toxic: In Agatha Christies murder stories the murderers take their victim’s breath with ‘sparkling cyanide’: a bit of innocent looking white powder potassium cyanide mixed into sparkling wine. Plants produce this poison with the help of enzymes when the plant is chewed.

Flora and fauna have developed a plethora of strategies to defend themselves from being eaten. Some run fast, others are spiky or simply taste disgusting. Some stone fruit, almond trees and ferns defend their young buds against feeding pests with cyanide. This poison expels the greatest enemy. This is due to an enzyme called hydroxynitrile lyase (HNL), which can release molecularly stored hydrogen cyanide. Luckily for the biotech industry, the enzyme can also catalyze the reverse reaction: it binds the cyanide to different molecules. This creates a double benefit. On the one hand, it is possible to recycle unwanted cyanide wastes. On the other hand, we can produce valuable building blocks for pharmaceutical agents, vitamin synthesis and other chiral products.

Smell enzyme acitivity

The researchers ‘sniff-screened’ a lot of different plants for an Amaretto-like smell. This worked by rubbing leaves or blossoms between the fingers. A smell of hydrocyanic acid and benzaldehyde can be perceived when the desired enzyme is there. From local forests and commercially available plants, finally a serpent fern was identified as a promising source of a new HNL enzyme. The fern’s name is white rabbit’s foot fern due to its white, fuzzy rhizomes that resemble a rabbit’s paw.

The amino acid sequence of the enzyme was elucidated by combining transcriptome analysis with LCMS/MS data of fern samples. Therefore, the sequences of all the expressed proteins present in the active plant were determined. In parallel, active protein samples were enriched and subjected to tryptic digestion and mass fingerprinting of the peptide fragments. The MS/MS data was used to search the transcriptome data. The new enzyme was extremely active, although it has not yet been optimized. To understand the fern HNL in depth, its 3D structure was examined. A comprehensive biochemical characterization was carried out to understand the enzymes scope and limitations.

The fern HNL is more efficient and simpler to handle than those previously known and used, because it is a small, uncomplicated enzyme that can be produced very well in the prokaryotic host Escherichia coli but also in the eukaryotic host Komagataella phaffii (formerly known as Pichia pastoris). These results are a perfect basis for the industrial utilization of this enzyme. Potential applications range from crop protection to the production of repellents or active pharmaceutical ingredients.