Repair of oxidized purines and damaged pyrimidines by E. coli Fpg protein: Different roles of proline 2 and lysine 57 residues

The Escherichia coli Fpg protein is involved in the repair of oxidized purines, including the highly mutagenic 7,8‐dihydro‐8‐oxoguanine (8‐oxoG). The Fpg protein also excises various oxidized pyrimidines with high efficiency. We examined, by targeted mutagenesis, the role of two highly conserved amino acid residues, proline 2 (P2) and lysine 57 (K57), on the catalytic activities of the Fpg protein toward a ring‐fragmentation product of thymine (αRT) and 5,6‐dihydrothymine (dHT). The following E. coli mutant Fpg proteins were investigated: lysine 57 → glycine (FpgK57G), proline 2 → glycine (FpgP2G), and proline 2 → glutamic acid (FpgP2E). The FpgK57G protein had barely detectable αRT and dHT‐DNA glycosylase activities and produced minute amounts of a Schiff‐base complex upon reaction with αRT containing DNA. In contrast, the activity of an FpgP2G mutant toward αRT was comparable to the wild type activity and produced a Schiff‐base complex with this substrate. FpgP2E was completely inactive in all the assays, in contrast, to the other mutants. The crystal structure of a homologous Fpg protein from an extreme thermophile, Thermus thermophilus HB8, reveals that it is composed of two distinct domains connected by a flexible hinge (Sugahara et al. [2000]: EMBO J 19:3857–3869). The N‐terminal proline, one primary residue for enzymatic catalysis, is positioned at the bottom of a cleft in close proximity to lysine 52 (analogous to K57 of the E. coli Fpg). Based on the biochemical assays, together with the crystal structure of T. thermophilus HB8 Fpg protein, we propose a two‐nucleophile model for the enzymatic catalysis. Environ. Mol. Mutagen. 39:10–17, 2002 © 2002 Wiley‐Liss, Inc.