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. 2014 Oct:89:32-44.
doi: 10.1016/j.toxicon.2014.06.020. Epub 2014 Jul 4.

A thermoactive L-amino acid oxidase from Cerastes cerastes snake venom: purification, biochemical and molecular characterization

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A thermoactive L-amino acid oxidase from Cerastes cerastes snake venom: purification, biochemical and molecular characterization

Zaineb Abdelkafi-Koubaa et al. Toxicon. 2014 Oct.

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.

Keywords: Glycosylation; Snake venom; Tertiary model; cDNA sequence; l-amino acid oxidase.

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