Cloning and biochemical characterization of APIT, a new l-amino acid oxidase from Aplysia punctata.
The purple ink of the sea hare Aplysia punctata contains a 60 kDa protein with tumoricidal activity. This A. punctata ink toxin (APIT) kills tumor cells within 6--8h in an apoptosis independent manner by the production of high amounts of hydrogen peroxide which induce a necrotic form of oxidative stress. Here, we describe the biochemical features of APIT associated with its anti-tumor activity. APIT is a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H(2)O(2)), ammonia (NH(4)(+)) and the corresponding alpha-keto acids. The tumoricidal effect is completely abrogated in the absence of the amino acids L-lysine and L-arginine. The enzyme is stable at temperatures from 0 to 50 degrees C. Similar to other FAD-binding enzymes, it is resistant against tryptic digest. Even digest with proteinase K fails to degrade the enzyme. Cloning of the APIT gene and subsequent sequencing revealed a FAD-binding domain followed by a so-called GG-motif, which is typical for L-amino acid oxidases. Strongest homology exists to escapin, aplysianin A precursor, the cyplasins L and S and achacin.
Butzke
Daniel
D
Department of Molecular Biology, Max Planck Institute for Infection Biology, Schumannstr. 21/22, D-10117 Berlin, Germany.
Hurwitz
Robert
R
Thiede
Bernd
B
Goedert
Sigrid
S
Rudel
Thomas
T
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000633:toxicity; D001048:Aplysia / Q000737:chemistry / Q000201:enzymology*; D016923:Cell Death; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D019286:Endopeptidase K / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D019169:Jurkat Cells; D050830:L-Amino Acid Oxidase / Q000138:chemical synthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000201:enzymology*; D009336:Necrosis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry
D000970:Antineoplastic Agents; D018076:DNA, Complementary; D006023:Glycoproteins; D007202:Indicators and Reagents; D008978:Mollusk Venoms; D050830:L-Amino Acid Oxidase; D014357:Trypsin; D019286:Endopeptidase K