Isolation and characterization of the first phosphodiesterase (Bj-PDE) from the venom of Bothrops jararacussu snake

• Published in: International journal of biological macromolecules Vol. 235; p. 123793
• Main Authors: Amorim, Fernanda Gobbi, Silva, Thiago Abrahão, de Oliveira Almeida, Gabriela, Redureau, Damien, Cabral, Hamilton, Quinton, Loïc, Sampaio, Suely Vilela
• Format: Journal Article Web Resource
• Language: English
• Published: Netherlands Elsevier B.V 30-04-2023
• Subjects: Animals; Biochemistry; Bothrops; Bothrops jararacussu; Chemistry; Chimie; Chromatography; Crotalid Venoms; Crotalid Venoms - chemistry; Edetic Acid; General Medicine; Molecular Biology; Native toxin; Phosphodiesterase; Phosphoric Diester Hydrolases; Phosphoric Diester Hydrolases - chemistry; Physical, chemical, mathematical & earth Sciences; Physique, chimie, mathématiques & sciences de la terre; Purification; Structural Biology; Venom; Bothrops jararacussu; Purification; Native toxin; Venom; Phosphodiesterase
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2023.123793

Phosphodiesterases are exonucleases that sequentially hydrolyse phosphodiester bonds of polynucleotides from the 3′-end and release 5-mononucleotides. After more than one decade without any advance in the study of Bothropic phosphodiesterases, we described here the isolation of the first phosphodiesterase from Bothrops jararacussu, which we named Bj-PDE. A five-step column chromatography procedure (size exclusion, hydrophobic interaction, cation exchange, lentil lectin affinity, and blue sepharose affinity) enabled isolation of Bj-PDE with preserved and stable enzymatic activity (bis(p-nitrophenyl) phosphate substrate), Km = 6.9 mM (± 0.7 mM), kcat/Km = 1.7 × 104 M−1 s−1 (± 0.2 × 104 M−1 s−1), MW = 116 kDa (SDS-PAGE), optimum activity around 45 °C at pH 8.0, and stability for 81 days at different storage temperatures (8, −20, and − 80 °C). Ca2+ and Mg2+ ions positively influenced Bj-PDE activity, while EDTA had the opposite action. Zn2+ restored >50 % of enzyme activity after its inhibition by EDTA. The Bj-PDE partial sequence identified by mass spectrometry was very similar to the sequence of BATXPDE1 from Bothrops atrox, which was evolutionarily close to this new PDE. Therefore, our study represents an important progress on the isolation of this minor toxin and sheds new lights on the properties and bioprospection of bothropic phosphodiesterases.
•This is the first report on isolation and characterization of one phosphodiesterase from B. jararacussu venom, named Bj-PDE.•Bj-PDE was purified after five chromatographic steps and presented stable enzymatic activity.•This 116 kDa enzyme was positively influenced by divalent ions and inhibited by EDTA.•Mass spectrometry confirmed that the partial sequence of Bj-PDE was structurally close to BATXPDE1 from Bothrops atrox.