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

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 f...

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Bibliographic Details
Published in:	Toxicon (Oxford) Vol. 139; pp. 74 - 86
Main Authors:	Lazo, Fanny, Vivas-Ruiz, Dan E., Sandoval, Gustavo A., Rodríguez, Edith F., Kozlova, Edgar E.G., Costal-Oliveira, F., Chávez-Olórtegui, Carlos, Severino, Ruperto, Yarlequé, Armando, Sanchez, Eladio F.
Format:	Journal Article
Language:	English
Published:	England Elsevier Ltd 01-12-2017
Subjects:	Amino Acid Sequence Animals Anti-Infective Agents - pharmacology Bacteria - drug effects Bothrops Bothrops pictus Bpic-LAAO Crotalid Venoms - enzymology Crotalid Venoms - toxicity Female Humans L-amino acid oxidase L-Amino Acid Oxidase - antagonists & inhibitors L-Amino Acid Oxidase - chemistry L-Amino Acid Oxidase - isolation & purification Male Mice Peru Phylogeny Platelet Aggregation - drug effects Structure-Activity Relationship Peru LAAO RP-HPLC CFU FAD PNGase F svLAAO Bpic-LAAO OVA Bothrops pictus L-amino acid oxidase
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Summary:	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 present a structure-function relationships of an L-amino acid oxidase, Bpic-LAAO, from Bothrops pictus.•Bpic-LAAO is a dimeric glycoprotein and its sequence was deduced from its cDNA.•Bpic-LAAO reduced the growth of P. aeruginosa, V. cholerae, S. aureus, E. faecalis and E. coli.
ISSN:	0041-0101 1879-3150
DOI:	10.1016/j.toxicon.2017.10.001