[en] 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.
Precision for document type :
Review article
Disciplines :
Chemistry
Author, co-author :
Gobbi Amorim, Fernanda ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Silva, Thiago Abrahão; Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n°, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address: thiago@fcfrp.usp.br
de Oliveira Almeida, Gabriela; Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n°, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address: gabi.almeida@usp.br
Redureau, Damien ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Cabral, Hamilton; Department of BioMolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil. Electronic address: hamilton@fcfrp.usp.br
Quinton, Loïc ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique
Sampaio, Suely Vilela; Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n°, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address: suvilela@usp.br
Language :
English
Title :
Isolation and characterization of the first phosphodiesterase (Bj-PDE) from the venom of Bothrops jararacussu snake.
Publication date :
30 April 2023
Journal title :
International Journal of Biological Macromolecules
CNPq - National Council for Scientific and Technological Development
Funding text :
We thank Mrs. Nanou Tanteliarisoa Haingo and Mrs. Lisette Trzpiot from the MSLab (ULiège) for their technical support in sample preparation, as well as to Mr. Maximilien Fléron and Mrs. Dominique Baiwir for their technical assistance in mass spectrometry analysis at the GIGA-Proteomics Facility (ULiège, EDRF funding). This study was supported by The National Council for Scientific and Technological Development, Brazil (CNPq – PQ 2018, grant #305282/2018-2). Q-Exactive mass spectrometer was supported by European Regional Development Fund (ERDF) and the Walloon Region grants, and Peaks X+ version Studio 10.5 software was supported by ERDF grant: BIOMED HUB Technology Support (number 2.2.1/996). F.G.A. performed the structural characterization by mass spectrometry and analysed data together with L.Q. and T.A.S. G.O.A. purified the protein and assessed its biological activities. T.A.S. analysed data and plotted the graphs. D.R. performed the phylogeny analysis. F.G.A and T.A.S wrote the paper. L.Q. and S.V.S. gave technical support. S.V.S. and H.C. devised the research project and supervised the experiments. S.V.S. searched for funding and revised the final manuscript. All authors read and approved the final manuscript.This study was supported by The National Council for Scientific and Technological Development , Brazil (CNPq – PQ 2018, grant # 305282/2018-2 ). Q-Exactive mass spectrometer was supported by European Regional Development Fund (ERDF) and the Walloon Region grants, and Peaks X+ version Studio 10.5 software was supported by ERDF grant: BIOMED HUB Technology Support (number 2.2.1/996 ).
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