One enzyme, many faces: urease is also canatoxin.

Area of Science:

• Biochemistry
• Plant Science
• Enzymology

Background:

• Ureases are enzymes that hydrolyze urea. Jack bean urease (JBU) is a hexameric protein. Canatoxin (CNTX) is a related neurotoxic protein isolated from jack bean seeds.
• CNTX exhibits lower molecular mass and enzyme activity than JBU and tends to form inactive oligomers upon storage.

Purpose of the Study:

• To characterize CNTX as an isoform of JBU.
• To elucidate the mechanism of CNTX stabilization by formaldehyde treatment.

Main Methods:

• Nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for peptide analysis.
• De novo sequencing to identify amino acid substitutions.
• MS/MS analysis of formaldehyde-treated CNTX.

Main Results:

• CNTX shares 804 identical amino acids with JBU (840 residues), indicating it is an isoform likely from a paralog gene.
• Fifteen peptides with amino acid substitutions were identified in CNTX.
• Formaldehyde treatment modified amino acid residues at the trimer-trimer interface of the JBU hexamer structure in CNTX.
• Stabilization prevents hexamer formation and aggregation.

Conclusions:

• CNTX is confirmed as an isoform of JBU.
• Formaldehyde stabilizes CNTX by modifying surface residues at the trimer-trimer interface, preventing oligomerization and preserving toxicity.