Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase

Raymond C. Manuel, Kenichi Hitomi, Andrew S. Arvai, Paul G. House, Andrew J. Kurtz, M. L. Dodson, Amanda K. McCullough, John A. Tainer, R. Stephen Lloyd

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary β-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-Å structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/ apyrimidinic lyase chemistry. The β-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the E142A or K20A mutation still catalyzes β and β-δ elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.

Original languageEnglish (US)
Pages (from-to)46930-46939
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number45
DOIs
StatePublished - Nov 5 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Manuel, R. C., Hitomi, K., Arvai, A. S., House, P. G., Kurtz, A. J., Dodson, M. L., McCullough, A. K., Tainer, J. A., & Lloyd, R. S. (2004). Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase. Journal of Biological Chemistry, 279(45), 46930-46939. https://doi.org/10.1074/jbc.M403944200