Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom

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Lazo F.
Vivas-Ruiz D.E.
Sandoval G.A.
Rodríguez E.F.
Kozlova E.E.G.
Costal-Oliveira F.
Chávez-Olórtegui C.
Severino R.
Yarlequé A.
Sanchez E.F.
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An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca2+, Mg2+ and Mn2+ did not alter Bpic-LAAO activity; however, Zn2+ is an inhibitor. Some reagents such as β-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 μg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus.
We thank Prof. J.A. Eble, from the Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Germany for critical reading the manuscript. This work was supported by Convenio de Cooperación Bilateral CONCYTEC (Perú) - CNPq (Brazil), Grant 490269/2013-3 , Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil) and Programa Nacional de Innovación para la Competitividad y Productividad - Innóvate Perú (Contrato N° 131-FINCyT-IB-2013 ). This report is part of a dissertation for Doctoral Degree of Fanny Lazo to Post Graduate School in Biological Sciences, UNMSM.
Palabras clave
L-aminoácido oxidasa, Ambosrops pictus, Bpic -LAAO