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

• Published in: Toxicon (Oxford) Vol. 89; pp. 32 - 44
• Main Authors: Abdelkafi-Koubaa, Zaineb, Jebali, Jed, Othman, Houcemeddine, Morjen, Maram, Aissa, Imen, Zouari-Kesentini, Raoudha, Bazaa, Amine, Ellefi, Amen Allah, Majdoub, Hafedh, Srairi-Abid, Najet, Gargouri, Youssef, El Ayeb, Mohamed, Marrakchi, Naziha
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.2014; Elsevier
• Subjects: Amino Acid Sequence; Amino acids; Base Sequence; cDNA sequence; Cerastes; Chromatography; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzymes; Glycosylation; Kinetics; l-amino acid oxidase; L-Amino Acid Oxidase - chemistry; L-Amino Acid Oxidase - isolation & purification; Life Sciences; Molecular Sequence Data; Optimization; Oxidase; Proteins; Sequence Alignment; Sequence Analysis, Protein; Snake venom; Snakes; Substrate Specificity; Tertiary model; Toxicology; Tryptophan; Viper Venoms - chemistry; cDNA sequence; NAT; BCA; Snake venom; OPD; CC-LAAO; l-amino acid oxidase; FAD; PNGase F; Glycosylation; SDS-PAGE; Tertiary model; SV-LAAOs; L -amino acid oxidase
• ISSN: 0041-0101; 1879-3150; 0041-0101
• DOI: 10.1016/j.toxicon.2014.06.020

A new l-amino acid oxidase (LAAO) from Cerastes cerastes snake venom, named CC-LAAO, was purified to homogeneity using a combination of size-exclusion, ion-exchange and affinity chromatography. CC-LAAO is a homodimeric glycosylated flavoprotein with a molecular mass around 58 kDa under reducing conditions and about 115 kDa in its native form when analyzed by SDS-PAGE and gel filtration chromatography, respectively. This enzyme displayed a Michaelis–Menten behavior with an optimal pH at 7.8. However, unlike known SV-LAAOs which display their maximum activity at 37 °C, CC-LAAO has an optimal temperature at 50 °C. Kinetic studies showed that the enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrates were L-Phe, L-Met and L-Leu. CC-LAAO activity was inhibited by the substrate analog N-acetyl tryptophan. The N-terminal amino acid sequence of this protein was determined by automated Edman degradation. The CC-LAAO cDNA was cloned from the venom gland total RNA preparation. The cDNA sequence contained an open-reading frame (ORF) of 1551-bp, which encoded a protein of 516 amino acids comprising a signal peptide of 18 amino acids and 498-residues mature protein. CC-LAAO sequence and its tertiary model shared high similarity with other snake venom LAAOs. •CC-LAAO is the first thermo-active SV-LAAOs which displayed an optimal activity at 50 °C.•Deduced amino acids sequence and tertiary model structure of CC-LAAO are performed.•CC-LAAO has five potential N-glycosylation sites and the sugar portion is not crucial for its enzymatic activity.