Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein

The Escherichia coil Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic Cg-oxo-gu anine (8-oxoG). The catalytic mechanism is believed to involve the formatio n of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importa nce and the role of this proline upon the various catalytic activities of t he Fpg protein was examined by targeted mutagenesis, resulting in the const ruction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E). The formamidopyrimidine DNA glycosyl ase activities of FpgP2G and FpgP2T were comparable and accounted for 10% o f the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DN A 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. FpgP2 E was completely inactive in all the assays. The binding constants of the d ifferent mutants when challenged with a duplex DNA containing a tetrahydrof uran residue were comparable. The mutant Fpg proteins barely or did not com plement in vivo the spontaneous transitions G/C --> T/A in E, coil BH990 (f pg 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 ac tivity.