Isolation and Structural Characterization of a Cytotoxic L-Amino Acid Oxidase from Agkistrodon contortrix laticinctus Snake Venom: Preliminary Crystallographic Data

• Published in: Archives of biochemistry and biophysics 1999-08, Vol.368 (2), p.285-290
• Main Authors: Souza, Dulce H.F., Eugenio, Luiz M., Fletcher, Jeffrey E., Jiang, Ming-Shi, Garratt, Richard C., Oliva, Glaucius, Selistre-de-Araujo, Heloisa S.
• Format: Article
• Language: English
• Subjects: Agkistrodon; Amino Acid Oxidoreductases - chemistry; Amino Acid Oxidoreductases - isolation & purification; Amino Acid Oxidoreductases - toxicity; Animals; apoptosis; Apoptosis - drug effects; Crotalid Venoms - enzymology; crystal structure; cytotoxicity; DNA Fragmentation - drug effects; HL-60 Cells; Humans; L-Amino Acid Oxidase; Protein Conformation; snake venom
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1287

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a Mr of 60,000 for the monomer as estimated by SDS–PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 μg/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris–HCl, pH 8.5, and 2 M (NH4)2SO4. Diffraction data collected to 3.5 Å showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 Å, c = 183.45 Å. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.