Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis.
Combining sequence analysis, structure prediction, and site-directed mutagenesis, we have investigated the mechanism of catalysis and substrate binding by the apoptotic mitochondrial nuclease EndoG, which belongs to the large family of DNA/RNA non-specific betabetaalpha-Me-finger nucleases. Catalysis of phosphodiester bond cleavage involves several highly conserved amino acid residues, namely His143, Asn174, and Glu182 required for water activation and metal ion binding, as well as Arg141 required for proper substrate binding and positioning, respectively. These results indicate that EndoG basically follows a similar mechanism as the Serratia nuclease, the best studied representative of the family of DNA/RNA non-specific nucleases, but that differences are observed for transition state stabilisation. In addition, we have identified two putative DNA/RNA binding residues of bovine EndoG, Arg135 and Arg186, strictly conserved only among mammalian members of the nuclease family, suggesting a similar mode of binding to single and double-stranded nucleic acid substrates by these enzymes. Finally, we demonstrate by ectopic expression of active and inactive variants of bovine EndoG in HeLa and CV1-cells that extramitochondrial active EndoG by itself induces cell death, whereas expression of an enzymatically inactive variant does not.
Schäfer
Patrick
P
Institute of Biochemistry, Justus-Liebig-University, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Scholz
Sebastian R
SR
Gimadutdinow
Oleg
O
Cymerman
Iwona A
IA
Bujnicki
Janusz M
JM
Ruiz-Carrillo
Adolf
A
Pingoud
Alfred
A
Meiss
Gregor
G
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001120:Arginine / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002417:Cattle; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009696:Nucleic Acids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D009696:Nucleic Acids; D011993:Recombinant Fusion Proteins; D001120:Arginine; D004706:Endodeoxyribonucleases; C052391:endonuclease G