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Background: MutY initiated base excision repair (BER) protects against mutations that otherwise result from oxidative damage to guanine. The 8-oxo-7,8-dihydro-guanine lesion (OG) pairs equally well with C or A, a source of ambiguity that explains high rates of GC → TA mutations for biallelic defects in the gene encoding MUTYH in humans. MutY/MUTYH intercepts the A:OG lesion and removes the chemically correct but informationally defective adenine nucleobase. A catalytic Glu – Glu43 in Geobacillus stearothermophilus MutY (Gs MutY); Glu37 in Escherichia coli MutY (Ec MutY); Glu134 in human MUTYH – is a defining chemical motif for A:OG adenine DNA glycosylases that is not found in other helix-hairpin-helix family members. While the importance of Glu for MutY’s catalytic mechanism is well understood, its origin is unknown. Discovery: Here we tested the structural and kinetic consequences of Glu replacement and found an alternate, slower mechanism, that yields a different stereoisomer for the AP site product. The impact on catalytic rate was consistent removal of acid/base catalysis and retention of transition state stabilization. High-resolution structures for E43Q and E43S substitution variants of Gs MutY show substrate disengagement and the enzyme-generated AP product in its alpha-anomer configuration. These results suggest that acquisition of Glu was a key innovation for emergence of the MutY/MUTYH lineage from an ancestral DNA glycosylase.
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Structural Basis for Nucleobase Activation by the Adenine DNA Glycosylase MutY
MutY excises adenine (A) from 8-oxo-guanine:adenine (OG:A) lesions in DNA to initiate base excision repair (BER) and thereby prevent mutations. A catalytic Glu, found at position 43 in the enzyme fromGeobacillus stearothermophilus(GsMutY), protonates the nucleobase at N7to labilize the N-glycosidic bond. The resulting oxocarbenium ion transition state is stabilized by a covalent DNA-enzyme intermediate and resolved by nucleophilic attack to yield thebeta-anomer abasic AP site product. The retaining SN1 mechanism for MutY posits deprotonation of the nucleophile by the catalytic Glu. Here we tested kinetic and structural consequences of Glu replacement and found that E43Q and E43S substitution variants were severely impaired, retained measurable activity, but engage the substrate nucleobase in ananticonformation, rotated by 180° from thesynconformation seen in previous substrate complexes. The enzyme-generated AP product is observed in itsalpha-anomer configuration for these Glu-replacement variants. Comparison with inverting adenine glycosylases that act on RNA or nucleosides shows that MutY's mechanism is uniquely reliant on one catalytic residue for both leaving group and nucleophile activation, a situation that may serve to ensure only rare adenines paired with OG are excised.
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- PAR ID:
- 10661019
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Posted Content:
- https://doi.org/10.64898/2026.01.22.701053
