The mechanism of the glycosylase reaction with hOGG1 base-excision repair enzyme: concerted effect of Lys249 and Asp268 during excision of 8-oxoguanine

Abstract

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.

Structural Insight into the Discrimination between 8-Oxoguanine Glycosidic Conformers by DNA Repair Enzymes: A Molecular Dynamics Study of Human Oxoguanine Glycosylase 1 and Formamidopyrimidine-DNA Glycosylase

“…In addition to broadening our understanding of how different OG glycosidic conformers are accommodated in the hOgg1 and FPG active sites, our simulations provide structural information about the DNA–enzyme prereaction complexes. Although QM/MM mechanistic studies are required to fully understand the reaction mechanism, our structural data sheds light on an existing controversy in the literature regarding the order of the first repair steps catalyzed by bifunctional DNA glycosylases. − ,− ,− Specifically, two S N 2 mechanisms have been previously proposed (Scheme ), , which differ in whether deglycosylation or sugar ring opening occurs upon nucleophilic attack (Lys249 in hOgg1 or Pro2 in FPG). Regardless of the OG conformation, the protonation state of Asp268 (hOgg1) or Glu3 (FPG), or the enzyme considered, the ∠(Nζ/N–C1′–O4′) nucleophilic angle corresponding to ring opening is closer to the ideal 180° expected for an S N 2 reaction than the ∠(Nζ/N–C1′–N9) angle corresponding to deglycosylation in the DNA–enzyme reactant complex.…”

“…Computational studies have provided a wealth of information about hOgg1 − and FPG. ,,− Previous molecular dynamics (MD) simulations on hOgg1 have compared the eversion of anti- OG and G from the DNA duplex into the active site to understand discrimination against G, examined the dynamics of wild-type (WT) and mutant hOgg1 bound to DNA containing anti- OG to gain insight into the role of active site residues and considered the potential recognition and catalysis mechanisms . Nevertheless, to the best of our knowledge, the ability of the hOgg1 active site to accommodate different OG glycosidic conformations has not been addressed.…”

“…In addition to the ambiguity surrounding the substrate binding orientation, the chemical reactions catalyzed by hOgg1 and FPG have not been completely explained despite computational studies investigating the enzymatic mechanisms using truncated models − ,, or complete DNA–enzyme complexes. ,− ,,, Two S N 2 mechanisms have been proposed for the first chemical step catalyzed by these enzymes. In the first mechanism, − ,,, nucleophilic attack (Lys249 in hOgg1 and Pro2 in FPG, Figure ) at C1′ of 2′-deoxyribose leads to base departure (deglycosylation, Scheme , left).…”

DNA Photodamage and Repair: Computational Photobiology in Action

“…The use of QM/MM algorithms has also allowed achieving some clear insights into DNA repair mechanisms, in particular obtaining highly precise and statistically converged free energy profile for the enzymatic reactions, also including photoactivated mechanisms in the case of bacterial systems [98][99][100][101][102]. However, less systematic studies are devoted to the global structural deformations induced by the different classes of lesions on the DNA and their consequence on the repair efficiency, as well as to the coupling between cluster lesions and their effects on the recognition by the repair machinery.…”

Computational studies of DNA repair: Insights into the function of monofunctional DNA glycosylases in the base excision repair pathway

“…To complement several reviews that have focused on experimental studies of BER enzymes (see, for example, references ) and to highlight key information provided from computer modeling, this Focus Article provides a mini‐review of computational studies of the chemical step facilitated by a selection of monofunctional DNA glycosylases. Although we acknowledge that several important computational works have examined the function of bifunctional DNA glycosylases (such as hOgg1, FPG and NEIL1), we focus herein on the challenge associated with hydrolysis of DNA glycosidic bonds. Furthermore, we emphasize the contributions of computational chemistry in deciphering the chemical step facilitated by several glycosylases, while recognizing that the base flipping step is also an important function of these repair enzymes and other computational work has shed light on this aspect of BER, including why some glycosylases do not base flip (AlkD) .…”