Catalytic Contributions of Key Residues in the Adenine Glycosylase MutY Revealed by pH-dependent Kinetics and Cellular Repair Assays

Abstract

Summary MutY enzymes prevent mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenines opposite OG. pH dependence analyses of the adenine glycosylase activity establish that Asp 138 of MutY must be deprotonated for maximal catalytic activity consistent with the role of this residue in stabilizing the oxacarbenium ion transition state in an SN1 mechanism. Use of a cellular OG:A repair assay allowed further validation of the critical role of Asp 138. Conservative substitutions of the catalytic residues Asp 138 and Glu 37 resulted in enzymes with a range of activity that were used to correlate the efficiency of adenine excision with overall OG:A repair and suppression of DNA mutations in vivo. The results indicate that MutY variations that reduce glycosylase activity as a consequence of reduced mismatch affinity result in more dramatic reductions in cellular OG:A repair than those that only compromise adenine excision catalysis.

Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine

“…Indeed, a truncated form of E. coli ( Ec ) MutY lacking the C-terminal domain exhibits reduced glycosylase activity, and lacks enhanced affinity for 8-oxoG containing duplexes compared to the WT enzyme [74, 81]. Moreover, the absence of the C-terminal domain with MutY diminishes the repair of an 8-oxoG:A plasmid substrate within a cellular context [82]. Notably, despite the ability of MutY enzymes to catalyze adenine removal from G:A and 8-oxoG:A substrates in vitro , minimal cellular repair on G:A-containing plasmid substrates was observed, again pointing to the critical nature of 8-oxoG recognition for selecting appropriate contexts for adenine excision [71].…”

“…The FLRC structure of Gs MutY (Figure 3C), along with structural, kinetic and mutagenesis studies, suggest that a Glu residue (E43 in Gs MutY, E120 in MUTYH) participates in catalysis as a general acid, while an Asp residue (D144 in Gs MutY, D222 in MUTYH) stabilizes the build-up of positive charge in the oxacarbenium ion intermediate [69, 77, 78, 82, 99]. The recently published structure of Gs MutY bound to an azaribose transition state analog (TSAC) provides a definitive picture of the key active site players necessary for glycosidic bond hydrolysis [15].…”

“…The active site Glu 43 is positioned to activate the water molecule for nucleophilic attack at the C1’ carbon of the acetal intermediate forming the β-anomer of the final abasic site product [15]. Both the Glu and Asp residues have been found to be essential for catalysis, and when these residues are mutated to remove the carboxylate (such as to Gln or Asn, respectively) the enzyme activity is completely abolished [69, 77, 82, 99]. Interestingly when the Glu residue was replaced with an Asp, the activity was severely diminished, yet when the Asp was converted to a larger Glu residue, the enzyme retained WT-like activity.…”

Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA

“…47 A conserved Asp side chain, which is essential for base excision, contacts adenine N7, suggesting a role in general acid catalysis of adenine expulsion. 48–50 MutY activity is reduced at least 10 4.4 -fold when N7 of adenine is replaced by carbon; it cannot excise 7-deaza-A. 47, 51 Moreover, activity is reduced 40-fold for 1-deaza-A, 100-fold for 3-deaza-A, 47, 52 and 6000-fold for an adenine analogue lacking both N1 and N3, 47, 53 suggesting that electrostatic contacts to N1 and N3 stabilize the departing adenine in the transition-state.…”

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase

“…In enzyme-catalyzed reactions, MutY, a DNA repair glycosylase involved in removal of adenine from G/A base pairs, catalyzes the excision of adenine by protonating the N7 position (49,50). Biochemical and structural studies identify a Glu residue (Glu 37 in E. coli MutY and Glu 43 in Bacillus stearothermophilus MutY) in the active site that can serve as a general acid in promoting N7 protonation (51)(52)(53)(54)(55). The close proximity of a water molecule between the Glu 43 in B. stearothermophilus MutY and N7 indicates that the proton for N7 protonation can come from water coordinated by Gu 43 (56).…”