Mechanism of action of base release by Escherichia coli Fpg protein: role of lysine 155 in catalysis.
Fpg protein (formamidopyrimidine/8-oxoguanine DNA N-glycosylase) is a DNA repair enzyme that catalyzes the removal of oxidized purines, most notably the mutagenic 7-hydro-8-oxoguanine (8oxoGua) lesion, by an N-glycosylase action. Additionally, Fpg protein catalyzes beta and delta elimination reactions subsequent to removal of the base lesions, as well as the analogous chemistry at abasic sites (AP sites). In this report, we show that of the two lysines that are conserved among the various putative prokaryotic Fpg proteins, a site specific alteration in one of them (lysine 155 changed to alanine) displays meaningful changes in substrate activities. However, lysine 155 is not required for the postulated covalent enzyme-substrate imine intermediate as demonstrated by trapping of the mutant protein-oligonucleotide complexes with cyanide or cyanoborohydride. The K155A mutant shows a decrease in activity with the 8oxoGua-substrate of approximately 50-fold under both k(cat)/Km and k(cat) conditions. This mutant also displays a similar reduction in activity with an oligonucleotide substrate possessing a single 2'-deoxy-8-oxonebularine site. In contrast, activity for a site specific 7-methylformamidopyrimidine-modified oligonucleotide is reduced approximately 3-4-fold, a much more modest decrease in activity. Interestingly, there is a concomitant increase in AP lyase activity above wild-type for the K155A mutant (1.6-fold increase in k(cat), 32-fold increase in k(cat)/Km), demonstrating retention of functional beta and delta lyase activities. Together these observations are readily accommodated by a model requiring a direct interaction of lysine 155 with the C8 oxygen of 8-oxopurines. Thus, conservation of this amino acid residue during evolution appears to be essential for specific incision of the mutagenic 8oxoGua base lesion by Fpg protein.
Rabow
L E
LE
Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia 30335, USA.
Kow
Y W
YW
D000595:Amino Acid Sequence; D002384:Catalysis; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008239:Lysine / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D012966:Sodium Cyanide / Q000378:metabolism
D029968:Escherichia coli Proteins; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D008239:Lysine; D012966:Sodium Cyanide