title: Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom.
abstract: The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K(m) of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 μg of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC₅₀: 22.7 μg/mL) and on MCF-7 cell line (breast adenocarcinoma, IC₅₀:1.41 μg/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC₅₀: 2.22 μg/mL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions.
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto-SP, Brazil.
authors.forename: Cristiane
authors.initials: C
authors.lastname: Bregge-Silva
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Nonato
authors.forename: Sérgio
authors.initials: S
authors.lastname: de Albuquerque
authors.forename: Paulo Lee
authors.initials: PL
authors.lastname: Ho
authors.forename: Inácio L M
authors.initials: IL
authors.lastname: Junqueira de Azevedo
authors.forename: Marcelo Ribeiro
authors.initials: MR
authors.lastname: Vasconcelos Diniz
authors.forename: Bruno
authors.initials: B
authors.lastname: Lomonte
authors.forename: Alexandra
authors.initials: A
authors.lastname: Rucavado
authors.forename: Cecilia
authors.initials: C
authors.lastname: Díaz
authors.forename: José María
authors.initials: JM
authors.lastname: Gutiérrez
authors.forename: Eliane Candiani
authors.initials: EC
authors.lastname: Arantes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D003435:Crotalid Venoms / Q000201:enzymology*; D006801:Humans; D050830:L-Amino Acid Oxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D007892:Leishmania braziliensis / Q000187:drug effects; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data
chemical_list: D000970:Antineoplastic Agents; D003435:Crotalid Venoms; C072644:Lachesis venom; D050830:L-Amino Acid Oxidase