Cloning and characterization of an antibacterial l-amino acid oxidase from Crotalus durissus cumanensis venom

• Published in: Toxicon (Oxford) 2013-03, Vol.64, p.1-11
• Main Authors: Vargas, Leidy Johana, Quintana, Juan Carlos, Pereañez, Jaime Andrés, Núñez, Vitelbina, Sanz, Libia, Calvete, Juan
• Format: Article
• Language: English
• Subjects: Acinetobacter - drug effects; Acinetobacter - growth & development; Acinetobacter - ultrastructure; Acinetobacter baumannii; Amino Acid Sequence; amino acids; Animals; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - isolation & purification; Anti-Bacterial Agents - pharmacology; Antibacterial activity; Cell Line; Cell Survival - drug effects; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cloning, Molecular; complementary DNA; Crotalid Venoms - enzymology; Crotalid Venoms - genetics; Crotalus - metabolism; Crotalus durissus; Crotalus durissus cumanensis; cytotoxicity; DNA, Complementary - metabolism; dose response; Escherichia coli; Humans; ion exchange; L-Amino Acid Oxidase - chemistry; L-Amino Acid Oxidase - genetics; L-Amino Acid Oxidase - isolation & purification; L-Amino Acid Oxidase - pharmacology; LAAO; Leukocytes, Mononuclear - drug effects; Mice; Microbial Sensitivity Tests; minimum inhibitory concentration; Molecular Sequence Data; molecular weight; mononuclear leukocytes; myoblasts; Myoblasts - drug effects; Oxidation-Reduction; Protein Structure, Tertiary; Sequence Analysis, DNA; Snake venom; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staphylococcus aureus; Staphylococcus aureus - drug effects; Staphylococcus aureus - growth & development; Staphylococcus aureus - ultrastructure; venoms
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2012.11.027

An l-amino acid oxidase (LAAO) from Crotalus durissus cumanensis venom (CdcLAAO) was purified to homogeneity using a combination of size-exclusion and ion exchange chromatographies. CdcLAAO is a monomeric protein exhibiting an apparent molecular mass of 55 kDa and a calculated pI of 8. Its complete 498-amino-acid sequence was deduced through cDNA and protein sequencing. The enzyme oxidized l-Leu with Km and a VMax of 9.23 μM and 0.46 μM/min respectively, and exhibited Kcat and a Kcat/Km of 1.8 s−1 and 195 mM−1s−1. CdcLAAO inhibited in a dose-dependent manner the growth of Staphylococcus aureus and Acinetobacter baumannii. The inhibitory effect was more significant on S. aureus, with a Minimal Inhibitory Concentration (MIC) of 8 μg/mL and Minimal Bactericidal Concentration (MBC) of 16 μg/mL, than against A. baumannii, with a MIC of 16 μg/mL and MBC of 32 μg/mL. However, against Escherichia coli CdcLAAO did not show inhibitory capacity at the concentrations tested (2–128 μg/mL). CdcLAAO did not exhibit cytotoxic activity on the mouse myoblast cell line C2C12 and on peripheral blood mononuclear cell (PBMC). ► Antibacterial activity of l-amino acid oxidase from Crotalus durissus cumanensis venom was detected. ► Complete 498-amino-acid sequence was deduced through cDNA and protein sequencing. ► CdcLAAO exhibited antimicrobial activity against Staphylococcus aureus and Acinetobacter baumanni but not against Escherichia coli.