Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity.
BACKGROUND
Endonuclease G (EndoG), a member of DNA/RNA nonspecific betabetaalpha-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.


METHODS
To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies.


RESULTS
Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified.


CONCLUSIONS
Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena.
Wu
Shih-Lu
SL
Department of Biochemistry, China Medical University, Taichung 40402, Taiwan. joyce@mail.cmu.edu.tw
Li
Chia-Cheng
CC
Chen
Jaw-Chyun
JC
Chen
Yi-Jin
YJ
Lin
Ching-Ting
CT
Ho
Tin-Yun
TY
Hsiang
Chien-Yun
CY
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019811:Hydroxylamine / Q000737:chemistry; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D000596:Amino Acids; D011994:Recombinant Proteins; D019811:Hydroxylamine; D004706:Endodeoxyribonucleases; C052391:endonuclease G; D008274:Magnesium