Reaction Intermediates in the Catalytic Mechanism of Escherichia coli MutY DNA Glycosylase

Abstract

The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary ␤-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-Å structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys 142 and Lys 20 . Analyses of the crystallographic structure also suggest a role for Glu 161 in the apurinic/ apyrimidinic lyase chemistry. The ␤-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes ␤ and ␤-␦ elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre-and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.Over the last 15 years, multiple laboratories have investigated the catalytic mechanism of DNA glycosylases that initiate the base excision repair (BER) 1 pathway (reviewed in Refs. 1-3). The elucidation of structure-activity relationships for DNA glycosylases and glycosylase/abasic site (AP) lyases has been facilitated both by solving the crystal structures and cocrystal complexes and analyses of chemical modification and trapping experiments (4 -17). These investigations have been successful in both localizing the active site pocket of these enzymes and identifying the key amino acids that participate in the catalytic events that lead to the excision of the inappropriate base. Determination of the chemical steps in the catalytic mechanism of DNA glycosylases is fundamental for understanding how organisms maintain their genome, despite the inevitable damage caused by exogenous and endogenous agents.DNA glycosylases with an associated AP lyase activity (␤-elimination) can be distinguished from DNA glycosylases without such activity, based on the identity of the nucleophile that attacks C1Ј of the deoxyribose sugar and whether the reaction proceeds through a covalent DNA-enzyme intermediate. DNA glycosylases that also catalyze a ␤-elimination reaction utilize a primary or secondary amine in the active site, whereas monofunctional glycosylases cleave the glycosyl bond via either the activation of a water molecule or a S N 1 attack (reviewed in Refs. 1 and 18). When a pr...

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

“…A factor that likely contributes to cell death is that BER of oxidative damage can be slow to complete because the lyase activities associated with MutM and MutY catalyze β or β,δ eliminations, thereby generating ends that require further processing to expose the 3′-OH needed for DNA polymerases (46). This DNA-based death mechanism, which initially can be counteracted by RecA-dependent or RecB/RecF-dependent processes, is likely complex.…”

DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

“…If this is a case, mutation of a seemingly critical residue often is not detrimental for at least some of the enzyme's functions to the extent that could be expected from the energy of the lost interactions, because the active site is flexible enough to compensate partially for the misfit. Among DNA glycosylases, E. coli MutYs have been shown to compensate for a loss of Lys-20, a residue partially accounting for its weak ␤-elimination activity, by using another active site amine, that of Lys-142 (42). In our own work with E. coli endonuclease VIII, 7 the mutations abolishing critical interactions with DNA phosphates and greatly diminishing the glycosylase activity have no effect on the AP lyase activity due to restructuring of the active site that fixes the substrate DNA in a catalytically competent conformation.…”

Role of a MutY DNA glycosylase in combating oxidative DNA damage in Helicobacter pylori

“…The H. pylori protein is slightly smaller, containing 328 amino acids versus 350 amino acids. H. pylori MutY also has the Glu37 and Asp 138 previously shown to make up the catalytic site of the E. coli enzyme (highlighted in figure 1B) [23]. Like in E. coli, H. pylori MutY has an endonuclease III domain, a helix-hairpin-helix domain, and an iron sulfur cluster.…”

“…The functions and mechanisms of E. coli MutY have been well studied [13,14,23,25,26,[28][29][30][31].…”