Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis.
Identifieur interne : 000013 ( Ncbi/Checkpoint ); précédent : 000012; suivant : 000014Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis.
Auteurs : M J Shroyer [États-Unis] ; S E Bennett ; C D Putnam ; J A Tainer ; D W MosbaughSource :
- Biochemistry [ 0006-2960 ] ; 1999.
Descripteurs français
- KwdFr :
- ADN (métabolisme), Catalyse, Chromatographie en phase liquide, Concentration en ions d'hydrogène, DNA Glycosylases, Escherichia coli (enzymologie), Liaison aux protéines, Mutagenèse dirigée, N-Glycosyl hydrolases (antagonistes et inhibiteurs), N-Glycosyl hydrolases (génétique), N-Glycosyl hydrolases (métabolisme), Sites de fixation, Spécificité du substrat, Séquence nucléotidique, Uracil-DNA glycosidase, Uracile (pharmacologie), Électrophorèse sur gel de polyacrylamide.
- MESH :
- antagonistes et inhibiteurs : N-Glycosyl hydrolases.
- enzymologie : Escherichia coli.
- génétique : N-Glycosyl hydrolases.
- métabolisme : ADN, N-Glycosyl hydrolases.
- pharmacologie : Uracile.
- Catalyse, Chromatographie en phase liquide, Concentration en ions d'hydrogène, DNA Glycosylases, Liaison aux protéines, Mutagenèse dirigée, Sites de fixation, Spécificité du substrat, Séquence nucléotidique, Uracil-DNA glycosidase, Électrophorèse sur gel de polyacrylamide.
English descriptors
- KwdEn :
- Base Sequence, Binding Sites, Catalysis, Chromatography, Liquid, DNA (metabolism), DNA Glycosylases, Electrophoresis, Polyacrylamide Gel, Escherichia coli (enzymology), Hydrogen-Ion Concentration, Mutagenesis, Site-Directed, N-Glycosyl Hydrolases (antagonists & inhibitors), N-Glycosyl Hydrolases (genetics), N-Glycosyl Hydrolases (metabolism), Protein Binding, Substrate Specificity, Uracil (pharmacology), Uracil-DNA Glycosidase.
- MESH :
- chemical , antagonists & inhibitors : N-Glycosyl Hydrolases.
- chemical , genetics : N-Glycosyl Hydrolases.
- chemical , metabolism : DNA, N-Glycosyl Hydrolases.
- enzymology : Escherichia coli.
- chemical , pharmacology : Uracil.
- Base Sequence, Binding Sites, Catalysis, Chromatography, Liquid, DNA Glycosylases, Electrophoresis, Polyacrylamide Gel, Hydrogen-Ion Concentration, Mutagenesis, Site-Directed, Protein Binding, Substrate Specificity, Uracil-DNA Glycosidase.
Abstract
The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated. Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung. The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues. Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0. Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer. However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate. Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil. Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP). Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D [Putnam, C. D., Shroyer, M. J. N., Lundquist, A. J., Mol, C. D., Arvai, A. S., Mosbaugh, D. W., and Tainer, J. A. (1999) J. Mol. Biol. 287, 331-346] provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage. A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.
DOI: 10.1021/bi982986j
PubMed: 10200172
Affiliations:
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pubmed:10200172Le document en format XML
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xml:lang="fr"><term>Catalyse</term><term>Chromatographie en phase liquide</term><term>Concentration en ions d'hydrogène</term><term>DNA Glycosylases</term><term>Liaison aux protéines</term><term>Mutagenèse dirigée</term><term>Sites de fixation</term><term>Spécificité du substrat</term><term>Séquence nucléotidique</term><term>Uracil-DNA glycosidase</term><term>Électrophorèse sur gel de polyacrylamide</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated. Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung. The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues. Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0. Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer. However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate. Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil. Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP). Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D [Putnam, C. D., Shroyer, M. J. N., Lundquist, A. J., Mol, C. D., Arvai, A. S., Mosbaugh, D. W., and Tainer, J. A. (1999) J. Mol. Biol. 287, 331-346] provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage. A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Bennett, S E" sort="Bennett, S E" uniqKey="Bennett S" first="S E" last="Bennett">S E Bennett</name><name sortKey="Mosbaugh, D W" sort="Mosbaugh, D W" uniqKey="Mosbaugh D" first="D W" last="Mosbaugh">D W Mosbaugh</name><name sortKey="Putnam, C D" sort="Putnam, C D" uniqKey="Putnam C" first="C D" last="Putnam">C D Putnam</name><name sortKey="Tainer, J A" sort="Tainer, J A" uniqKey="Tainer J" first="J A" last="Tainer">J A Tainer</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Shroyer, M J" sort="Shroyer, M J" uniqKey="Shroyer M" first="M J" last="Shroyer">M J Shroyer</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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