A thermoactive l-amino acid oxidase from Cerastes cerastes snake venom: Purification, biochemical and molecular characterization

Abdelkafi-Koubaa, Zaineb; Jebali, Jed; Othman, Houcemeddine; Morjen, Maram; Aissa, Imen; Zouari-Kesentini, R.; Bazaa, Amine; Ellefi, Amen Allah; Majdoub, Hafedh; Srairi-Abid, Najet; Gargouri, Youssef; Ayeb, Mohamed El; Marrakchi, Naziha

doi:10.1016/j.toxicon.2014.06.020

Cited by 19 publications

(16 citation statements)

References 41 publications

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“…The purified LAAO enzyme isoforms isolated in this study seems to be noncovalently linked heterodimeric glycoproteins unlike other snake venom LAAOs which are in general, homodimeric glycoproteins [16], [17], [21], [22], [23], they have molecular masses of 60, 56 kDa for LAAOI and 60, 53 kDa for LAAOII as measured by reducing electrophoresis. This result is also unlike that of Abdelkafi-Koubaa et al [24], who isolated a homodimeric single form of glycosylated flavoprotein LAAO from C. cerastes venom collected from Tunis, with a molecular mass around 58 kDa under reducing conditions and about 115 kDa in its native form. The heterogenicity obtained in our results may reflect the polymorphism of the C. cerastes LAAO enzymes among the donor snakes.…”

Section: Discussioncontrasting

confidence: 95%

“…The results in the present study revealed that both Cc-LAAO enzyme isoforms maintained their full activity at a wide range of pH from 4 to 9 and the optimum pHs were 7.8 and 7 for Cc-LAAOI and Cc-LAAOII, respectively. These results are similar to that of Abdelkafi-Koubaa et al [24]. Similar to all the reported venom LAAOs, the temperatures higher than 50 °C result in a gradual decrease in activity caused by disruptions in hydrophobic interactions and hydrogen bonds between the different subunits of the enzyme [24], [30].…”

Section: Discussionsupporting

confidence: 91%

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Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing

El-Hakim¹,

Salama²,

Hamed³

et al. 2015

Journal of Genetic Engineering and Biotechnology

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Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from Cerastes cerastes venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The Km and Vmax values were 0.67 mM, 0.135 μmol/min for LAAOI and 0.82 mM, 0.087 μmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn2+ ion markedly increased the LAAO activity for both purified isoforms, while Na+, K+, Ca2+, Mg2+ and Ba2+ ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn2+, Ni2+, Co2+, Cu2+ and AL3+ ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, β-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.

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Section: Discussioncontrasting

confidence: 95%

Section: Discussionsupporting

confidence: 91%

Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing

El-Hakim¹,

Salama²,

Hamed³

et al. 2015

Journal of Genetic Engineering and Biotechnology

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“…L-Leu is the most common substrate for SV-LAAOs; however, high catalytic constants (K cat /K m ) have also been described for L-Phe, L-Met and L-Ile [ 1 ]. According to the results obtained by Abdelkafi-Koubaa and collaborators [ 4 ], the LAAO from Cerastes cerastes venom presented the highest specificity for Phe, followed by Met and Leu. Similarly, the LAAO from Bothrops pirajai venom also presented the highest specificity for Phe, followed by Tyr and Trp [ 25 ].…”

Section: Resultsmentioning

confidence: 99%

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

Costa

Carone

Tucci

et al. 2018

J Venom Anim Toxins Incl Trop Dis

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BackgroundL-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms.Methods and resultsThe enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4 °C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU.ConclusionsOur results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.

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“…The assay was conducted in a 96‐well microplate according to Abdelkafi‐Koubaa et al . Briefly, in each well, 90 μL of the substrate solution containing 250 mM of l ‐arginine, 2 mM O‐phenylenediamine, and 0.81 U/mL of horseradish peroxidase was mixed with 10 μL of the enzyme.…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73‐like, from Cerastes cerastes venom

Saoud

Chérifi

Benhassine

et al. 2016

J Biochem & Molecular Tox

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The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid sequence of Cc-5'NTase displayed high homology with many nucleotidases. Its activity was optimal at pH 7 with a specific hydrolytic activity toward mono-, di-, and triphosphate adenylated nucleotides. Cc-5'NTase preferentially hydrolyzed ADP and obeyed Michaelis-Menten kinetics. Among the metals and inhibitors tested, Ni and Mg completely potentiated enzyme activity, whereas EGTA, PMSF, iodoacetamide, vanillic acid, vanillyl mandelic acid, and 1,10-phenanthroline partially abolished its activity. Cc-5'NTase was not lethal for mice at 5 mg/kg and exhibited in vivo anticoagulant effect. It also dose-dependently inhibited adenosine diphosphate-induced platelet aggregation by converting adenosine diphosphate to adenosine and prohibited arachidonic acid-induced aggregation but was not effective on fibrinogen-induced aggregation. Cc-5'NTase could be a good tool as pharmacological molecule in thrombosis diagnostic and/or therapy.

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A thermoactive l-amino acid oxidase from Cerastes cerastes snake venom: Purification, biochemical and molecular characterization

Cited by 19 publications

References 41 publications

Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing

Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73‐like, from Cerastes cerastes venom

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