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 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.
Souza
D H
DH
Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP, 13565-905, Brazil.
Eugenio
L M
LM
Fletcher
J E
JE
Jiang
M S
MS
Garratt
R C
RC
Oliva
G
G
Selistre-de-Araujo
H S
HS
D017836:Agkistrodon; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D003435:Crotalid Venoms / Q000201:enzymology*; D053938:DNA Fragmentation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D050830:L-Amino Acid Oxidase; D011487:Protein Conformation
D003435:Crotalid Venoms; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase