Structural snapshots of base excision by the cancer-associated variant MutY N146S reveal a retaining mechanism

Abstract

DNA glycosylase MutY plays a critical role in suppression of mutations resulted from oxidative damage, as highlighted by cancer-association of the human enzyme. MutY requires a highly conserved catalytic Asp residue for excision of adenines misinserted opposite 8-oxo-7,8-dihydroguanine (OG). A nearby Asn residue hydrogen bonds to the catalytic Asp in structures of MutY and its mutation to Ser is an inherited variant in human MUTYH associated with colorectal cancer. We captured structural snapshots of N146S Geobacillus stearothermophilus MutY bound to DNA containing a substrate, a transition state analog and enzyme-catalyzed abasic site products to provide insight into the base excision mechanism of MutY and the role of Asn. Surprisingly, despite the ability of N146S to excise adenine and purine (P) in vitro, albeit at slow rates, N146S-OG:P complex showed a calcium coordinated to the purine base altering its conformation to inhibit hydrolysis. We obtained crystal structures of N146S Gs MutY bound to its abasic site product by removing the calcium from crystals of N146S-OG:P complex to initiate catalysis in crystallo or by crystallization in the absence of calcium. The product structures of N146S feature enzyme-generated β-anomer abasic sites that support a retaining mechanism for MutY-catalyzed base excision.

Distinctive Formation of a DNA–Protein Cross-Link during the Repair of DNA Oxidative Damage: Insights into Human Disease from MD Simulations and QM/MM Calculations

“…N146 was also implicated as a modulator of catalytic activity via hydrogen bonding with D144, which was recently verified by a measured ∼180-fold decrease in adenine excision upon N146S mutation . However, NMR experiments on the MutY methanolysis product and a crystal structure of MutY bound to the enzyme-generated product revealed a β-anomer abasic site with retention of stereochemistry at C1′, which contrasts with the inversion of stereochemistry associated with the previously proposed , and computationally studied , catalytic pathways, as well as the accepted mechanisms for any other monofunctional DNA glycosylase. These structural and kinetic data necessitated a revised mechanism in which D144 directly attacks the oxocarbenium ion in an irreversible reaction step to yield a DNA–protein cross-link (Figure C).…”

“…Owing to the same identities and conformations of active site catalytic residues, MUTYH is expected to share a catalytic mechanism with the Escherichia coli adenine DNA glycosylase homologue (MutY). , Although the function of MutY has been subjected to many experimental studies, − the deglycosylation mechanism is highly debated in the literature. Mutational data, kinetic isotope effect (KIE) analysis, and a crystal structure of 8oG:A bound to the D144N mutant of Geobacillus stearothermophilus ( Gs ) MutY (known as the lesion recognition complex (LRC)) led to the first adopted mechanism (Figure A) .…”

“…Cross-link formation is consistent with the observed short distance (2.9 Å) between the carboxylate group of D144 and N1′ (the azaribose atom equivalent to C1′ of deoxyribose) and retention of stereochemistry at the anomeric carbon and would permit active site reorganization, including nucleobase release and alignment of an E43-activated water molecule to hydrolyze the cross-link and regenerate the enzyme in the final chemical step. Although compatible with crystallographic, − mutational, − , and KIE data, and parallels to mechanisms established for glycosidases, , a structural depiction of the DNA–protein cross-link and atomic level details of the cross-link formation mechanism to support the unique double-displacement mechanism for a DNA glycosylase is currently lacking.…”

Metagenome mining and functional analysis reveal oxidized guanine DNA repair at the Lost City Hydrothermal Field

“…indicating these chemical motifs stabilize the transition state during catalysis (32)(33)(34). For the LCHF MutYs, the Asn residue is invariant, the catalytic Asp is nearly invariant, replaced by chemically similar Glu for five LCHF MutYs, and the catalytic Tyr residue is often replace by Ser and sometimes by Thr or Asn.…”

“…Protein structures for the LCHF MutY representatives were predicted by the AlphaFold2 plugin within UCSF ChimeraX (33,34). Predicted structures were superimposed with crystal structure of Gs MutY (pdb 3G0Q) using the MatchMaker tool in ChimeraX to generate RMSD (Å) for pruned atom pairs.…”