Uracil DNA Glycosylase: Revisiting Substrate-Assisted Catalysis by DNA Phosphate Anions

Abstract

Uracil DNA glycosylase (UNG) is a powerful DNA repair enzyme that has been shown to stabilize a glycosyl cation reaction intermediate and a related tight binding inhibitor using electrostatic interactions with the +1 and −1, but not the +2, phosphodiester group of the single-stranded DNA substrate Ap 2+ Ap 1+ Up 1− ApA. These experimental results differed considerably from computational findings using duplex DNA, where the +2 phosphate was found to stabilize the transition state by ~5 kcal/mol, suggesting that UNG uses different catalytic strategies with single-stranded and doublestranded DNA substrates. In addition, the computational studies indicated that the conserved and positively charged His 148 (which hydrogen bonds to the +2 phosphate) destabilized the glycosyl cation intermediate by 6-8 kcal/mol through anticatalytic electrostatic interactions. To evaluate these interesting proposals, we measured the kinetic effects of neutral methylphosphonate (MeP) stereoisomers at the +1 and +2 positions of a 12-mer dsDNA substrate and also the catalytic contribution and ionization state of His 148. For MeP substitutions at the +1 position, single-turnover kinetic studies showed that the activation barrier was increased by 9.8 and 3.1 kcal/mol, corresponding to a stereoselectivity of nearly 40000-fold for the respective MeP isomers. Identical to the findings with ssDNA, MeP substitutions at the +2 position resulted in only small changes in the activation barrier (±0.3 kcal/mol), with little stereoselectivity (~4-fold). However, the H148A mutation destabilizes both the ground state and transition states by 2.4 and 4.3 kcal/mol, respectively. Thus, His 148 is catalytic because it stabilizes the transition state to a greater extent (1.9 kcal/mol) than the ground state. Heteronuclear NMR studies established that His 148 was neutral in the free enzyme at neutral pH, and in conformational exchange in a specific DNA complex containing uracil. We conclude that the +1 and +2 phosphate esters play identical catalytic roles in the reactions of single-stranded and double-stranded DNA substrates, and that His 148 serves a catalytic role by positioning the substrate and catalytic water, or by an environmental effect.The diverse functional groups of substrates can provide unique opportunities for enzymes to co-opt mechanisms of catalysis that may not be possible in the absence of these groups. One example is the polymeric and negatively charged DNA phosphate ester backbone that can provide a robust scaffold for electrostatically driven binding interactions for enzymes that recognize DNA substrates (1). Quite often, such interactions may involve noncovalent bonding at one or more phosphate sites distant from the site of chemical reaction, and the favorable binding energy is used to drive the substrate into a reactive state that would be otherwise energetically inaccessible. More remarkably, the DNA repair enzyme uracil DNA glycosylase

Crystal Structure of Human Methyl-Binding Domain IV Glycosylase Bound to Abasic DNA

“…29, 31, 32 It may also facilitate the chemical step, by helping to position the anionic phosphates to stabilize the transient glycosyl cation intermediate; such interactions account for up to 50% of the rate enhancement provided by UDG. 33-35 Notably, for MBD4, the distances between C1′ of the flipped nucleotide and the putative catalytic phosphates (+1, 5.6 Å; −1, 4.9 Å; −2, 8.5 Å) are similar to those observed for UDG 31, 34 (+1, 5.5 Å; −1, 5.0 Å; −2, 9.9 Å), suggesting that such electrostatic interactions could also be important for the chemical step of the MBD4 reaction.…”

The substrate binding interface of alkylpurine DNA glycosylase AlkD

“…Alternatively, inhibition of AlkD catalyzed 7mG excision by methylphosphonate substitution suggests that the distorted DNA backbone conformation may allow a phosphate in close proximity to the lesion to provide electrostatic stabilization or nucleophilic activation through a substrate-assisted mechanism of catalysis [15], as previously observed for uracil excision from DNA by UDG [11, 16, 17]. Such a mechanism would suggest that without contacts to the lesion, AlkD must rely on enzyme-substrate binding energy to distort the DNA backbone into an autocatalytic conformation to promote base excision.…”

Depurination of N7-Methylguanine by DNA Glycosylase AlkD Is Dependent on the DNA Backbone

“…4,7,16–18 The best studied example is human uracil DNA glycosylase, in which DNA phosphates promote glycosidic bond cleavage by stabilizing the charge or conformation of the oxocarbenium intermediate. 4,7,16,17,19–21 …”