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 McCullough, John A. Tainer, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

45 Citations (Scopus)

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

Fingerprint

DNA Glycosylases
Reaction intermediates
Adenine
Escherichia coli
Lyases
Lysine
DNA
Catalytic Domain
Mutagenesis
Genomic Instability
Equilibrium constants
Guanine
Site-Directed Mutagenesis
Catalysis
Plasticity
Crystal structure
Maintenance
X-Rays
X rays
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase. / Manuel, Raymond C.; Hitomi, Kenichi; Arvai, Andrew S.; House, Paul G.; Kurtz, Andrew J.; Dodson, M. L.; McCullough, Amanda; Tainer, John A.; Lloyd, Robert (Stephen).

In: Journal of Biological Chemistry, Vol. 279, No. 45, 05.11.2004, p. 46930-46939.

Research output: Contribution to journalArticle

Manuel, RC, Hitomi, K, Arvai, AS, House, PG, Kurtz, AJ, Dodson, ML, McCullough, A, Tainer, JA & Lloyd, RS 2004, 'Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase', Journal of Biological Chemistry, vol. 279, no. 45, pp. 46930-46939. https://doi.org/10.1074/jbc.M403944200
Manuel, Raymond C. ; Hitomi, Kenichi ; Arvai, Andrew S. ; House, Paul G. ; Kurtz, Andrew J. ; Dodson, M. L. ; McCullough, Amanda ; Tainer, John A. ; Lloyd, Robert (Stephen). / Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 45. pp. 46930-46939.
@article{b09761e416f446a895dd14ce7a6d0213,
title = "Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase",
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-{\AA} 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.",
author = "Manuel, {Raymond C.} and Kenichi Hitomi and Arvai, {Andrew S.} and House, {Paul G.} and Kurtz, {Andrew J.} and Dodson, {M. L.} and Amanda McCullough and Tainer, {John A.} and Lloyd, {Robert (Stephen)}",
year = "2004",
month = "11",
day = "5",
doi = "10.1074/jbc.M403944200",
language = "English (US)",
volume = "279",
pages = "46930--46939",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "45",

}

TY - JOUR

T1 - Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase

AU - Manuel, Raymond C.

AU - Hitomi, Kenichi

AU - Arvai, Andrew S.

AU - House, Paul G.

AU - Kurtz, Andrew J.

AU - Dodson, M. L.

AU - McCullough, Amanda

AU - Tainer, John A.

AU - Lloyd, Robert (Stephen)

PY - 2004/11/5

Y1 - 2004/11/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=8744274358&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=8744274358&partnerID=8YFLogxK

U2 - 10.1074/jbc.M403944200

DO - 10.1074/jbc.M403944200

M3 - Article

C2 - 15326180

AN - SCOPUS:8744274358

VL - 279

SP - 46930

EP - 46939

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 45

ER -