Oxidative DNA damage is generated by a variety of environmental and endogenous agents, including ionizing radiation, certain chemicals, and products of aerobic metabolism (1). 8-oxoG 1 is one of the most abundant forms of oxidative DNA damage (2). Due to its ability to form a Hoogstein-type base pair with adenine (3), 8-oxoG is miscoding (4) and mutagenic, resulting in G3 T transversions in bacterial and eukaryotic cells (5, 6). The potential harmful effects of this lesion are avoided by base excision repair. In Escherichia coli, formamidopyrimidine-DNA glycosylase (Fpg, EC 3.2.2.23) removes 8-oxoG, Me-FaPy, and several structurally related lesions from damaged DNA (7,8). Fpg is a component of the "GO system" that includes MutY, a mismatch adenine-DNA glycosylase, and MutT, an 8-oxodGTPase (9, 10); E. coli strains deficient in any of these genes are strong mutators (11).Fpg shares significant sequence homology with endonuclease VIII (Nei) of E. coli (12). Both proteins belong to a family unrelated by sequence or tertiary structure to a larger family of DNA glycosylases, for which the prototype is endonuclease III (Nth) (13,14). The substrate specificity of Fpg differs significantly from Nei (7, 8, 15) but closely resembles that of the eukaryotic 8-oxoguanine-DNA glycosylase, Ogg1, a member of the Nth family (14,16,17). Fpg also possesses AP lyase activity, nicking the phosphodiester backbone of DNA at the site of the lesion. Base excision by Fpg is followed immediately by two ␤-elimination steps, resulting in a single nucleotide gap flanked by phosphate termini (7). A Schiff base intermediate, involving Pro-1 of the enzyme and C1Ј of the damaged nucleotide, forms early in the reaction sequence and can be reductively trapped by treatment with NaBH 4 forming a stable covalent complex (18,19). The mechanism of cleavage is similar to that of Nei (15,20), but not to that of Ogg1 where only one ␤-elimination occurs, and the efficiency of the elimination step is very low compared with base excision (16,17).Comparing the structures of Fpg, Nei, and Ogg1 provides a unique opportunity to analyze features of damage recognition and catalysis common to DNA glycosylases/AP lyases. The presence of DNA enhances the analytic power of the model by revealing the precise nature of enzyme-DNA interactions. The structure of the human Ogg1 catalytic domain complexed to DNA has been solved (21, 22), as has the structure of E. coli Nei covalently cross-linked to DNA by NaBH 4 (23). The structure of Fpg from Thermus thermophilus HB8 (Tth-Fpg) has recently been solved in the absence of DNA (24). Although mechanisms for lesion recognition and catalysis by Fpg have been suggested on the basis of this structure and on earlier biochemical studies of E. coli Fpg (8,18,24,25), many questions remain unanswered regarding the mode of Fpg-DNA interactions and the catalytic reaction mechanism of this important DNA repair protein.To investigate the mechanisms of Fpg-DNA interactions, we have utilized NaBH 4 reduction of the Schiff base intermediate t...

contributed equally to this work Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential b-elimination steps. However, Nei differs signi®cantly from Fpg in substrate speci®city. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A Ê resolution. The crosslink is derived from a Schiff base intermediate that precedes b-elimination and is stabilized by reduction with NaBH 4 . Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identi®ed. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.

Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O 8 of extrahelical 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.Oxidative metabolism produces reactive oxygen species that damage cellular DNA. DNA bases, deoxyribose, and the cellular nucleoside triphosphate pool are prone to such events. Redox reactions result in modification of the canonical DNA bases, with formamidopyrimidine (FaPy)

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.