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Standard role for a conserved aspartate or more direct involvement in deglycosylation? An ONIOM and MD investigation of adenine-DNA glycosylase.

Biochemistry (2013-10-31)
Jennifer L Kellie, Katie A Wilson, Stacey D Wetmore

8-Oxoguanine (OG) is one of the most frequently occurring forms of DNA damage and is particularly deleterious since it forms a stable Hoogsteen base pair with adenine (A). The repair of an OG:A mispair is initiated by adenine-DNA glycosylase (MutY), which hydrolyzes the sugar-nucleobase bond of the adenine residue before the lesion is processed by other proteins. MutY has been proposed to use a two-part chemical step involving protonation of the adenine nucleobase, followed by SN1 hydrolysis of the glycosidic bond. However, differences between a recent (fluorine recognition complex, denoted as the FLRC) crystal structure and the structure on which most mechanistic conclusions have been based to date (namely, the lesion recognition complex or LRC) raise questions regarding the mechanism used by MutY and the discrete role of various active-site residues. The present work uses both molecular dynamics (MD) and quantum mechanical (ONIOM) models to compare the active-site conformational dynamics in the two crystal structures, which suggests that only the understudied FLRC leads to a catalytically competent reactant. Indeed, all previous computational studies on MutY have been initiated from the LRC structure. Subsequently, for the first time, various mechanisms are examined with detailed ONIOM(M06-2X:PM6) reaction potential energy surfaces (PES) based on the FLRC structure, which significantly extends the mechanistic picture. Specifically, our work reveals that the reaction proceeds through a different route than the commonly accepted mechanism and the catalytic function of various active-site residues (Geobacillus stearothermophilus numbering). Specifically, contrary to proposals based on the LRC, E43 is determined to solely be involved in the initial adenine protonation step and not the deglycosylation reaction as the general base. Additionally, a novel catalytic role is proposed for Y126, whereby this residue plays a significant role in stabilizing the highly charged active site, primarily through interactions with E43. More importantly, D144 is found to explicitly catalyze the nucleobase dissociation step through partial nucleophilic attack. Although this is a more direct role than previously proposed for any other DNA glycosylase, comparison to previous work on other glycosylases justifies the larger contribution in the case of MutY and allows us to propose a unified role for the conserved Asp/Glu in the DNA glycosylases, as well as other enzymes that catalyze nucleotide deglycosylation reactions.

Product Number
Product Description

L-Aspartic acid, BioXtra, ≥99% (HPLC)
L-Aspartic acid, reagent grade, ≥98% (HPLC)
L-Aspartic acid, BioUltra, ≥99.5% (T)
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L-Aspartic acid, Pharmaceutical Secondary Standard; Certified Reference Material
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