Computational Investigation of Glycosylase and β-Lyase Activity Facilitated by Proline: Applications to FPG and Comparisons to hOgg1

Abstract

Quantum mechanical methods are used to investigate the chemical steps during the bifunctional (glycosylase and β-lyase) activity of bacterial FPG DNA glycosylase, which removes the major oxidation product (8-oxoguanine) from DNA as part of the base excision repair process. To facilitate investigation of all potential pathways, the smallest chemically relevant model is implemented, namely a modified OG nucleoside-3'-monophosphate and a truncated proline nucleophile. Potential energy surfaces are characterized with SMD-M06-2X/6-311+G(2df,2p)//PCM-B3LYP/6-31G(d) and compared to a previous study on the analogues human enzyme (hOgg1), which uses a lysine nucleophile (Kellie, J. L.; Wetmore, S. D. J. Phys. Chem. B 2012, 116, 10786-10797). Our large calculated barriers indicate that FPG must actively catalyze the three main phases of the overall reaction, namely, deglycosylation, (deoxyribose) ring-opening, and β-elimination, and provide clues about how this is achieved through comparison to accurate crystal structures. The main conclusions about key mechanistic steps hold true regardless of the nucleophile, suggesting that most major differences in the relative activity of FPG and hOgg1 are primarily due to other active site residues. Nevertheless, support for possible monofunctional (deglycosylation only) activity is only evident when lysine is the nucleophile. This finding agrees with experimental observations of monofunctional activity of hOgg1 and further supports the broadly accepted bifunctional activity of FPG.

Molecular dynamics simulation of the opposite-base preference and interactions in the active site of formamidopyrimidine-DNA glycosylase

“…1b); quantum mechanics/molecular mechanics (QM/MM) simulations show that O4′ protonation provides a low-barrier path to glycosidic bond cleavage by Fpg and its eukaryotic functional analog, OGG1 [33, 36, 68]. From several structures Fpg–DNA complexes, it has been suggested that the proton is shuttled from N[Pro1] to Oε2[Glu2], perhaps through a network of crystallographic water molecules present in the active site [19, 26]; this possibility was also favored by QM/MM analysis [69]. However, no attempt to estimate p K a of Pro1 and Glu2 has been reported in the literature.…”

Quantum mechanical study of the β- and δ-lyase reactions during the base excision repair process: application to FPG

“…This component was built from the ring-opened deoxyribose intermediate optimized in our previous study 28 by replacing the 5 0 -methoxy group with a 5 0 -phosphate group. This component was built from the ring-opened deoxyribose intermediate optimized in our previous study 28 by replacing the 5 0 -methoxy group with a 5 0 -phosphate group.…”

“…[28][29][30][31][32][33][34][35] Previous computational work includes molecular dynamics simulations of the anti (w defined as +(O4 0 -C1 0 -N9-C4) equals 180 AE 901) and syn (w = 0 AE 901) conformers of OG, (unmodified) G and several other lesions (such as FapyG, 4,6-diamino-5-formamidopyrimidine (FapyA), and 7,8-dihydro-8-oxoadenine (8-oxoA)) bound in the FPG active site, which shed light on the lesion binding modes and substrate interactions in the recognition pocket. [28][29][30][31][32][33][34][35] Previous computational work includes molecular dynamics simulations of the anti (w defined as +(O4 0 -C1 0 -N9-C4) equals 180 AE 901) and syn (w = 0 AE 901) conformers of OG, (unmodified) G and several other lesions (such as FapyG, 4,6-diamino-5-formamidopyrimidine (FapyA), and 7,8-dihydro-8-oxoadenine (8-oxoA)) bound in the FPG active site, which shed light on the lesion binding modes and substrate interactions in the recognition pocket.…”

“…Specifically, (a) an active-site base abstracts pro-S-2 0 , which is followed by (b) the elimination of the 3 0 -phosphate. 28,38 However, the nucleoside-3 0 -monophosphate models employed in the previous studies were neutralized by a sodium ion, 28,38 which prevents the phosphate protonation that has been proposed to facilitate the reaction. In FPG, the 3 0 -phosphate has been proposed to be protonated by Lys57 and further stabilized by Arg259, while the 5 0 -phosphate has been proposed to be protonated by Arg259 and further stabilized by Tyr237 and Asn169.…”

“…In contrast to previous computational studies on the b-elimination step, 28,38 anionic phosphate models are used to investigate the role of phosphate activation in the elimination reactions. In contrast to previous computational studies on the b-elimination step, 28,38 anionic phosphate models are used to investigate the role of phosphate activation in the elimination reactions.…”