Heteronuclear NMR and Crystallographic Studies of Wild-Type and H187Q Escherichia coli Uracil DNA Glycosylase:  Electrophilic Catalysis of Uracil Expulsion by a Neutral Histidine 187

Abstract

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.

A Novel Zinc Snap Motif Conveys Structural Stability to 3-Methyladenine DNA Glycosylase I

“…NMR Spectroscopy-The NMR sample, prepared as described in a previous report (4), contained ϳ0.5 mM TAG, 10 mM phosphate buffer (pH 6.6), 100 mM NaCl, 3 mM dithiothreitol, 0.34 mM NaN 3 , and 10% D 2 O in a total volume of 300 l. The two-dimensional 1 H-15 N LR HSQC experiment was conducted at 20°C on a Varian Unity Plus 600-MHz spectrometer equipped with four frequency channels and pulse-field gradients as described (12). This experiment was simply a conventional HSQC used for backbone amide correlations collected with an optimized two-bond 2 J NH value of 22 Hz in order to observe signals from the weak two-bond couplings in the histidine rings and suppress the signals from the one-bond J NH amide couplings.…”

“…In the consensus sequences, an asterisk indicates identical amino acid residues and a colon indicates highly conserved residues. The metal-binding Cys 2 His 2 cluster, CX [12][13][14][15][16][17] HX n HX 3 C, is highlighted in black. The NCBI gene identification (GI) numbers of the various TAG sequences are shown next to the names of the species.…”

Turning On Uracil-DNA Glycosylase Using a Pyrene Nucleotide Switch

“…In addition, the pyrene rescue hypothesis predicts that other mutations of UDG that do not affect the baseflipping step would not be rescued by pyrene substitution. Two such mutations that have been previously investigated by our group are D64N, which removes the water-activating group, and H187G, which removes the catalytic electrophile (17,24,25). Thus, these mutant enzymes were also tested with the pyrene substrates.…”

Comment on "Uracil DNA Glycosylase Activity Is Dispensable for Immunoglobulin Class Switch"

“…To reconcile this contradictory result with that of the single mutants, the authors attributed it to Ba structural requirement for an unknown function of UNG. [ However, structural studies of the H268Q and D145N single mutants provide no evidence that structural perturbations arise from these mutations, and it would be unexpected if the double mutations differed (10,11). Structural studies clearly place the His268, Asn204, and Asp145 catalytic groups deep within the active site pocket of UNG, such that it is unlikely that they have any other role except in the catalytic process of uracil recognition and excision.…”