title: A thermoactive L-amino acid oxidase from Cerastes cerastes snake venom: purification, biochemical and molecular characterization.
abstract: 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.
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie. Electronic address: abdelkafi_zaineb@yahoo.fr.
authors.forename: Zaineb
authors.initials: Z
authors.lastname: Abdelkafi-Koubaa
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Jed
authors.initials: J
authors.lastname: Jebali
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Houcemeddine
authors.initials: H
authors.lastname: Othman
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Maram
authors.initials: M
authors.lastname: Morjen
authors.affiliation: Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Ecole Nationale d'Ingénieurs de Sfax (ENIS). Route de Soukra, BP 1173, 3038 Sfax, Tunisie.
authors.forename: Imen
authors.initials: I
authors.lastname: Aissa
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Raoudha
authors.initials: R
authors.lastname: Zouari-Kesentini
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Amine
authors.initials: A
authors.lastname: Bazaa
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Amen Allah
authors.initials: AA
authors.lastname: Ellefi
authors.affiliation: USCR Séquenceur de Protéines, Faculté des Sciences de Sfax, BP 1171 Route de Soukra 3000 Sfax, Tunisie.
authors.forename: Hafedh
authors.initials: H
authors.lastname: Majdoub
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Najet
authors.initials: N
authors.lastname: Srairi-Abid
authors.affiliation: Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Ecole Nationale d'Ingénieurs de Sfax (ENIS). Route de Soukra, BP 1173, 3038 Sfax, Tunisie.
authors.forename: Youssef
authors.initials: Y
authors.lastname: Gargouri
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Ayeb
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis, Tunisie; Faculté de Medecine de Tunis, 1007 Tunis, Tunisie.
authors.forename: Naziha
authors.initials: N
authors.lastname: Marrakchi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007700:Kinetics; D050830:L-Amino Acid Oxidase / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014757:Viper Venoms / Q000737:chemistry*
chemical_list: D014757:Viper Venoms; D050830:L-Amino Acid Oxidase
keywords: Glycosylation; Snake venom; Tertiary model; cDNA sequence; l-amino acid oxidase