Mechanistic and Conformational Flexibility of the Covalent Linkage Formed during β-Lyase Activity on an AP-Site: Application to hOgg1

The beta/delta-lyase activity of bifunctional glycosylases on damaged nucleotides in DNA involves the formation of a covalent linkage between the protein (lysine or N-terminal proline) and DNA (C1' of the damaged nucleotide). In the present study, the conformational and mechanistic flexibility of the cross-link is examined. Repair of 8-oxoguanine damage by hOgg1 is considered as a representative system, and the glycosylase through beta-lyase steps are investigated using density functional theory. (PCM/SMD)-M06-2X/6-311+G(2df,2p)//PCM-B3LYP/6-31G(d) energetics were determined for eight unique mechanisms differing in the conformation of the imine linkage (E/Z), the proton (pro-S/R) abstracted during elimination, and whether the ring-opening step is base catalyzed. This initial study used a model system limited to the damaged nucleoside 3'-monophosphate and a model nucleophile to investigate this series of complex reaction steps. The great flexibility exhibited by the linkage and clustered beta-elimination energetics indicate sterics will play a large role in predicting the preferred lyase mechanism for a given enzyme. The stationary points identified herein can be overlaid into a protein structure to assist in generating initial guesses for large model systems. By comparing the characterized geometries and enzyme active sites, methods for catalysis of the various chemical steps can be identified, and these possibilities are discussed in detail for hOgg1. Interestingly, the most stable structure on the potential energy surface occurs before elimination of the 3'-phosphate. Hydrolysis of the protein-DNA cross-link at this point would yield an AP-site, which provides support for the recently observed monofunctional activity of hOgg1.