Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase.
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 beta-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-A 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 beta-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 K142A or K20A mutation still catalyzes beta and beta-delta 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.
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
Raymond C
RC
Manuel
Kenichi
K
Hitomi
Andrew S
AS
Arvai
Paul G
PG
House
Andrew J
AJ
Kurtz
M L
ML
Dodson
Amanda K
AK
McCullough
John A
JA
Tainer
R Stephen
RS
Lloyd
D000225:Adenine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D045647:DNA Glycosylases / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D013997:Time Factors
D001224:Aspartic Acid; D018698:Glutamic Acid; D006147:Guanine; D004247:DNA; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D000225:Adenine; D008239:Lysine