Publications

• Apoxin I, a novel apoptosis-inducing factor with L-amino acid oxidase activity purified from Western diamondback rattlesnake venom.

  Torii S., Naito M., Tsuruo T.

  Venom of the western diamondback rattlesnake (Crotalus atrox) induces apoptosis in human umbilical vein endothelial cells, which could result in hemorrhage in tissues bitten by the snake. To identify the hemorrhagic factor, we purified a novel protein, apoxin I, from rattlesnake venom. Apoxin I in ... >> More

  Venom of the western diamondback rattlesnake (Crotalus atrox) induces apoptosis in human umbilical vein endothelial cells, which could result in hemorrhage in tissues bitten by the snake. To identify the hemorrhagic factor, we purified a novel protein, apoxin I, from rattlesnake venom. Apoxin I induced apoptosis in human umbilical vein endothelial, human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells. Amino acid sequence analysis of the apoxin I showed close similarity to L-amino acid oxidase from the Malayan pit viper (Calloselasma rhodostoma). The purified apoxin I oxidized L-leucine but not D-leucine to produce H2O2. The apoxin I-induced apoptosis was inhibited by catalase, a H2O2 scavenger. These results indicate that the H2O2 produced by L-amino acid oxidation by apoxin I is involved in the apoxin I-induced apoptosis and in hemorrhage caused by rattlesnake venom. << Less

  J. Biol. Chem. 272:9539-9542(1997) [PubMed] [EuropePMC]

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

  Souza D.H.F., Eugenio L.M., Fletcher J.E., Jiang M.-S., Garratt R.C., Oliva G., Selistre-de-Araujo H.S.

  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 ... >> More

  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 micrograms/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 (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase. << Less

  Arch. Biochem. Biophys. 368:285-290(1999) [PubMed] [EuropePMC]

• On the reaction mechanism of Crotalus adamanteus L-amino acid oxidase.

  Massey V., Curti B.

  J. Biol. Chem. 242:1259-1264(1967) [PubMed] [EuropePMC]

• [Kinetic studies on the reaction mechanism of L-amino acid oxidase from Neuraspora crassa].

  Luppa D., Aurich H.

  Acta Biol Med Ger 27:839-850(1971) [PubMed] [EuropePMC]

• Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity.

  Torii S., Yamane K., Mashima T., Haga N., Yamamoto K., Fox J.W., Naito M., Tsuruo T.

  We previously purified apoxin I, an apoptosis-inducing factor with L-amino acid oxidase (LAO) activity, from Western diamondback rattlesnake venom. To determine the primary structure of apoxin I, we cloned its cDNA. The amino acid sequence showed that apoxin I has an FAD binding domain and shares ... >> More

  We previously purified apoxin I, an apoptosis-inducing factor with L-amino acid oxidase (LAO) activity, from Western diamondback rattlesnake venom. To determine the primary structure of apoxin I, we cloned its cDNA. The amino acid sequence showed that apoxin I has an FAD binding domain and shares homology with L-amino acid oxidase (LAO) from Neurospora crassa, human monoamine oxidase B, and mouse interleukin 4-induced F1G1 protein. The full-length apoxin I has an N-terminal signal sequence that is processed in mature apoxin I in venom. When the apoxin I gene was transfected into human 293T cells, the recombinant protein was expressed in the cells, and a significant amount of apoxin I was secreted into the medium. The secreted recombinant apoxin I protein showed LAO and apoptosis-inducing activity, but the recombinant protein in the cells did not, suggesting that maturation and secretion of the apoxin I protein is needed for its activity. Treating the transfected cells with tunicamycin inhibited the secretion and LAO activity of the recombinant apoxin I. In addition, deleting the amino-terminal region flanking the signal sequence, the FAD-binding domain and the carboxy-terminal region abolished the secretion and LAO activity of the recombinant proteins. These results indicate that in order for apoxin I to become active, these regions and posttranslational modification, such as N-glycosylation, are required. << Less

  Biochemistry 39:3197-3205(2000) [PubMed] [EuropePMC]

• Crystal structure of LAAO from Calloselasma rhodostoma with an L-phenylalanine substrate: insights into structure and mechanism.

  Moustafa I.M., Foster S., Lyubimov A.Y., Vrielink A.

  L-Amino acid oxidase is a dimeric glycosylated flavoenzyme, a major constituent of the venom-from the snake Calloselasma rhodostoma. The enzyme exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities. The structure of l-amino acid oxidase with its substrate (L-phenylal ... >> More

  L-Amino acid oxidase is a dimeric glycosylated flavoenzyme, a major constituent of the venom-from the snake Calloselasma rhodostoma. The enzyme exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities. The structure of l-amino acid oxidase with its substrate (L-phenylalanine) has been refined to a resolution of 1.8 A. The complex structure reveals the substrate bound to the reduced flavin (FADred). Alternative conformations for the key residues His223 and Arg322 are evident, suggesting a dynamic active site. Furthermore, conformational changes are apparent for the isoalloxazine ring; the three-ring system exhibits more bending around the N5-N10 axis compared to the oxidized flavin. The implications of the observed dynamics on the mechanism of catalysis are discussed. Inspection of buried surfaces in the enzyme reveals a Y-shaped channel system extending from the external surface of the protein to the active site. One portion of this channel may serve as the entry path for O2 during the oxidative half-reaction. The second region, separated from the proposed O2 channel by the N terminus (residues 8-16) of the protein, may play a role in H2O2 release. Interestingly, the latter portion of the channel would direct the H2O2 product to the exterior surface of the protein, near the glycan moiety, thought to anchor the enzyme to the host cell. This channel location may explain the ability of the enzyme to localize H2O2 to the targeted cell and thus induce the apoptotic effect. << Less

  J. Mol. Biol. 364:991-1002(2006) [PubMed] [EuropePMC]