Scripps VIVO scripps research logo

  • Index
  • Log in
  • Home
  • People
  • Organizations
  • Research
  • Events
Search form

Mutation of an active site residue in escherichia coli uracil-DNA glycosylase: Effect on DNA binding, uracil inhibition and catalysis

Academic Article
uri icon
  • Overview
  • Identity
  • Additional Document Info
  • View All
scroll to property group menus

Overview

related to degree

  • Putnam, Christopher, Ph.D. in Biology, Scripps Research 1994 - 2000

authors

  • Shroyer, M. J. N.
  • Bennett, S. E.
  • Putnam, Christopher
  • Tainer, John
  • Mosbaugh, D. W.

publication date

  • April 1999

journal

  • Biochemistry  Journal

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.

subject areas

  • Base Sequence
  • Binding Sites
  • Catalysis
  • Chromatography, Liquid
  • DNA
  • DNA Glycosylases
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Hydrogen-Ion Concentration
  • Mutagenesis, Site-Directed
  • N-Glycosyl Hydrolases
  • Protein Binding
  • Substrate Specificity
  • Uracil
  • Uracil-DNA Glycosidase
scroll to property group menus

Identity

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi982986j

PubMed ID

  • 10200172
scroll to property group menus

Additional Document Info

start page

  • 4834

end page

  • 4845

volume

  • 38

issue

  • 15

©2021 The Scripps Research Institute | Terms of Use | Powered by VIVO

  • About
  • Contact Us
  • Support