Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom:: Preliminary crystallographic data

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 M-r 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 mu g/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)(2)SO4. Diffraction data collected to 3.5 Angstrom showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 Angstrom, c = 183.45 Angstrom. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase. (C) 1999 Academic Press.