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 K.
AU - Tainer, John A.
AU - Lloyd, R. 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.
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U2 - 10.1074/jbc.M403944200
DO - 10.1074/jbc.M403944200
M3 - Article
C2 - 15326180
AN - SCOPUS:8744274358
SN - 0021-9258
VL - 279
SP - 46930
EP - 46939
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 45
ER -