Preorganized Internal Electric Field Promotes a Double-Displacement Mechanism for the Adenine Excision Reaction by Adenine DNA Glycosylase

Abstract

Adenine DNA glycosylase (MutY) is a monofunctional glycosylase, removing adenines (A) misinserted opposite 8-oxo-7,8-dihydroguanine (OG), a common product of oxidative damage to DNA. Through multiscale calculations, we decipher a detailed adenine excision mechanism of MutY that is consistent with all available experimental data, involving an initial protonation step and two nucleophilic displacement steps. During the first displacement step, N-glycosidic bond cleavage is accompanied by the attack of the carboxylate group of residue Asp144 at the anomeric carbon (C1′), forming a covalent glycosyl-enzyme intermediate to stabilize the fleeting oxocarbenium ion. After departure of the excised base, water nucleophiles can be recruited to displace Asp144, completing the catalytic cycle with retention of stereochemistry at the C1′ position. The two displacement reactions are found to mostly involve the movement of the oxocarbenium ion, occurring with large charge reorganization and thus sensitive to the internal electric field (IEF) exerted by the polar protein environment. Intriguingly, we find that the negatively charged carboxylate group is a good nucleophile for the oxocarbenium ion, yet an unactivated water molecule is not, and that the electric field catalysis strategy is used by the enzyme to enable its unique double-displacement reaction mechanism. A strong IEF, pointing toward 5′ direction of the substrate sugar ring, greatly facilitates the second displacement reaction at the expense of elevating the barrier of the first one, thereby allowing both reactions to occur. These findings not only increase our understanding of the strategies used by DNA glycosylases to repair DNA lesions, but also have important implications for how internal/external electric field can be applied to modulate chemical reactions.

FSHing for DNA Damage: Key Features of MutY Detection of 8-Oxoguanine:Adenine Mismatches

“…In all three product complex structures, only the beta -anomer was observed, consistent with retention of configuration and aligning with the mechanism we had previously proposed. In addition, two recent computational studies have provided additional support for this mechanism that is unique to MutY relative to other glycosylases. , …”

“…In addition, two recent computational studies have provided additional support for this mechanism that is unique to MutY relative to other glycosylases. 36 , 37 …”

Designed Local Electric Fields─Promising Tools for Enzyme Engineering

“…Furthermore, local electric fields can be very heterogeneous, so that not all parts of the reacting system experience the same field strength, and the dynamics of both substrates at the active site, as well as of the protein matrix, can change the nature of the electrostatic interactions significantly throughout the reaction. , …”