The human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a bifunctional DNA repair enzyme that possesses both glycosylase and AP-lyase activity. Its AP-lyase reaction mechanism had been revealed by crystallographic capturing of the intermediate adduct. However, no intermediate within the glycosylase reaction was reported to date and the relevant reaction mechanism thus remained unresolved. In this work, we studied the glycosylase reaction of hOGG1 by time-resolved crystallography and spectroscopic/enzymological analyses. To trigger the glycosylase reaction within a crystal, we created a pH-responsive mutant of hOGG1 in which lysine 249 (K249) has been replaced by histidine (H), and designated hOGG1(K249H). Using hOGG1(K249H), a reactive intermediate state of the hOGG1(K249H)-DNA complex was captured in crystal upon pH activation. An unprecedented, ribose-ring-opened hemiaminal structure at the 8-oxoguanine (oxoG) site was found. Based on the structure of the reaction intermediate and QM/MM (quantum mechanics/molecular mechanics) calculations, a glycosylase reaction pathway of hOGG1(K249H) was identified where the aspartic acid 268 (D268) acts as a proton donor to O4' of oxoG. Moreover, enzymologically derived pKa (4.5) of a catalytic residue indicated that the observed pKa can be attributed to the carboxy group of D268. Thus, a reaction mechanism of the glycosylase reaction by hOGG1(K249H) has been proposed.

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key DNA repair enzyme that excises 8-oxoguanine, a mutagenic base lesion, from double-stranded DNA. In this study, we crystallographically visualized an intermediate state of the enzymatic reaction. To achieve this, we employed a specifically designed pH-sensitive mutant of hOGG1 and applied a freeze-trapping technique to capture the reaction intermediate. The resulting crystal structure revealed a previously unknown reaction pathway involving a hemiaminal-type intermediate, captured here for the first time. These findings provide new insights into the catalytic mechanism of hOGG1.

• MeSH
• DNA Glycosylases * chemistry   genetics   metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Guanine analogs & derivatives   chemistry   metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Mutation MeSH
• DNA Repair * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 8-hydroxyguanine MeSH Browser
• DNA Glycosylases * MeSH
• DNA MeSH
• Guanine MeSH
• oxoguanine glycosylase 1, human MeSH Browser

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.

• MeSH
• Biocatalysis MeSH
• DNA Glycosylases chemistry   metabolism   MeSH
• Guanine analogs & derivatives   metabolism   MeSH
• Aspartic Acid metabolism   MeSH
• Humans MeSH
• Lysine metabolism   MeSH
• Models, Molecular MeSH
• Mutant Proteins chemistry   metabolism   MeSH
• DNA Repair * MeSH
• Proton Magnetic Resonance Spectroscopy MeSH
• Thermodynamics MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 8-hydroxyguanine MeSH Browser
• DNA Glycosylases MeSH
• Guanine MeSH
• Aspartic Acid MeSH
• Lysine MeSH
• Mutant Proteins MeSH
• oxoguanine glycosylase 1, human MeSH Browser