Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein.
The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C --> T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity.
Sidorkina
O M
OM
Groupe "Réparation des Lésions Radio- et Chimio- Induites," UMR 8532 Centre National de la Recherche Scientifique, Institut Gustave Roussy, 39, Rue Camille Desmoulins, 94805 Villejuif Cédex, France.
Laval
J
J
D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011392:Proline; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D011392:Proline; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli