4EUG

Crystallographic and Enzymatic Studies of an Active Site Variant H187Q of Escherichia Coli Uracil DNA Glycosylase: Crystal Structures of Mutant H187Q and its Uracil Complex

Summary for 4EUG

• Entry DOI: 10.2210/pdb4eug/pdb
• Related: 2EUG 3EUG 5EUG
• Descriptor: PROTEIN (GLYCOSYLASE) (2 entities in total)
• Functional Keywords: glycosylase, hydrolase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P12295
• Total number of polymer chains: 1
• Total formula weight: 25696.12

Authors

Xiao, G.,Tordova, M.,Drohat, A.C.,Jagadeesh, J.,Stivers, J.T.,Gilliland, G.L. (deposition date: 1998-12-27, release date: 1999-07-23, Last modification date: 2023-09-13)

Primary citation

Drohat, A.C.,Xiao, G.,Tordova, M.,Jagadeesh, J.,Pankiewicz, K.W.,Watanabe, K.A.,Gilliland, G.L.,Stivers, J.T.
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.
Biochemistry, 38:11876-11886, 1999

PubMed 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.

PubMed: 10508390
DOI: 10.1021/bi9910880

Experimental method

X-RAY DIFFRACTION (1.4 Å)