The mechanism of the glycosylase reaction with hOGG1 base-excision repair enzyme: concerted effect of Lys249 and Asp268 during excision of 8-oxoguanine.
The excision of 8-oxoguanine (oxoG) by the human 8-oxoguanine DNA glycosylase 1 (hOGG1) base-excision repair enzyme was studied by using the QM/MM (M06-2X/6-31G(d,p):OPLS2005) calculation method and nuclear magnetic resonance (NMR) spectroscopy. The calculated glycosylase reaction included excision of the oxoG base, formation of Lys249-ribose enzyme-substrate covalent adduct and formation of a Schiff base. The formation of a Schiff base with ΔG# = 17.7 kcal/mol was the rate-limiting step of the reaction. The excision of the oxoG base with ΔG# = 16.1 kcal/mol proceeded via substitution of the C1΄-N9 N-glycosidic bond with an H-N9 bond where the negative charge on the oxoG base and the positive charge on the ribose were compensated in a concerted manner by NH3+(Lys249) and CO2-(Asp268), respectively. The effect of Asp268 on the oxoG excision was demonstrated with 1H NMR for WT hOGG1 and the hOGG1(D268N) mutant: the excision of oxoG was notably suppressed when Asp268 was mutated to Asn. The loss of the base-excision function was rationalized with QM/MM calculations and Asp268 was confirmed as the electrostatic stabilizer of ribose oxocarbenium through the initial base-excision step of DNA repair. The NMR experiments and QM/MM calculations consistently illustrated the base-excision reaction operated by hOGG1.
The Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo námestí 2, 166 10 Praha, Czech Republic.
Jakub
J
Šebera
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Nishihama-Boji 180, Yamashiro-cho, Tokushima 770 8514, Japan.
Yoshikazu
Y
Hattori
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 980 8578, Japan.
Daichi
D
Sato
Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Zámek 136, 373 33 Nové Hrady, Czech Republic.
David
D
Reha
The Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo námestí 2, 166 10 Praha, Czech Republic.
Radim
R
Nencka
Division of Genome Biology, National Cancer Center Research Institute 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104 0045, Japan.
Takashi
T
Kohno
Graduate School of Engineering, Yokohama National University, Tokiwadai 79-5, Hodogaya-ku, Yokohama 240 8501, Japan.
Chojiro
C
Kojima
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Nishihama-Boji 180, Yamashiro-cho, Tokushima 770 8514, Japan.|Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 980 8578, Japan.
Yoshiyuki
Y
Tanaka
The Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo náměstí 2, 166 10 Praha, Czech Republic.|Department of Electrotechnology, Electrical Engineering Czech Technical University, Technická 2, 166 27 Praha, Czech Republic.
Vladimír
V
Sychrovský
D001224:Aspartic Acid / Q000378:metabolism*; D055162:Biocatalysis; D045647:DNA Glycosylases / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D066244:Proton Magnetic Resonance Spectroscopy; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D050505:Mutant Proteins; D001224:Aspartic Acid; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D008239:Lysine