Fpg Protein

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Biochemistry of Fpg

Fpg (EC 3.2.2.23), also known as 8-oxoguanine-DNA glycosylase or MutM, is one of the most important enzymes in the bacterial BER pathway. Fpg protects DNA from the accumulation of 8-oxoguanine (8-oxoG), which tends to adopt an energetically favorable syn conformation, thereby forming a Hoogsteen-type base pair with adenine. The addition of dAMP opposite 8-oxoG during replication results in a G→T transversion. Fpg has several enzymatic activities. It acts as a glycosylase/AP lyase responsible for the removal of damaged bases, followed by cleavage of the DNA backbone at the site of damage by β-elimination.

But Fpg differs from most other glycosylases/AP lyases in its ability to carry out two consecutive-elimination steps (often called "β-elimination" and "δ-elimination" to distinguish them). Thus, the final product of Fpg action is a single nucleotide gap in DNA flanked by two phosphate groups. In addition, Fpg acts as a deoxyribophosphodiesterase-lyase, catalyzing the removal of deoxyribose-5’-phosphate from nicked abasic (AP) sites in DNA. A covalent imine (Schiff base) is formed between the enzyme and the C1’ of the damaged nucleotide as an intermediate in this catalytic reaction, which can be reduced with sodium borohydride to form a stable covalent Fpg-DNA complex. In addition to 8-oxoG and FaPy lesions, Fpg also excised many other modified purines and possibly some oxidized pyrimidines.

Structures of Fpg proteins from different bacterial species

Structural data for Fpg proteins from E. coli (Eco-Fpg), Lactococcus lactis, Bacillus stearothermophilus, and Thermus thermophilus are currently available. Th. thermophilus Fpg was resolved as an uncomplexed protein, while other Fpg proteins were analyzed bound to DNA, but these protein structures were overall quite similar.

Fpg consists of N-terminal and C-terminal domains connected by hinge polypeptides. The N-terminal domain contains the active site for Schiff base formation, in which Pro-1 acts as the nucleophile. The C-terminal domain contains a single zinc finger required for binding damaged DNA. Figure 1 presents a schematic representation of the Fpg-DNA interaction from X-ray data.

Similar to most other DNA glycosylases, Fpg everts the damaged nucleotide from DNA into the deep active site pocket. To recognize the orphaned, opposing cytosine residues (C⁽⁰⁾ ), the enzyme wedges the aryl ring of Phe-110 into the space between C⁽⁰⁾ and the adjacent base (C⁽¹⁾) and makes face-to-face interactions with the pyrimidine ring of C⁽¹⁾, thereby unstacking the two bases. C⁽⁰⁾ remains intra-helix and is stabilized by hydrogen bonds between C⁽⁰⁾ and O² and N3 of Arg-108.

Figure 1. Scheme of Fpg-DNA interactions (Zharkov, D.O. et al. 2003)

Contribution of structural elements of DNA to Fpg binding

The Fpg-induced nonspecific DNA adjustments to the catalytically optimal conformation could not be detected in stopped-flow experiments. For many DNA-dependent enzymes, this adjustment and subsequent catalytic steps are most sensitive to the nature of the DNA. Each enzyme fine-tunes the DNA conformation in its own way, often involving melting of DNA fragments, often accompanied by conformational changes in the DNA backbone, and partial or complete base unpacking. The formation of structurally defined hydrogen bonds between enzymes and specific DNA bases is one of the final stages of substrate selection. The formation of these bonds ensures the proper orientation of the specific bases in the enzyme's active site and speeds up the reaction by 4-7 orders of magnitude.

The increase in affinity at the transition from nonspecific to specific ds ODNs depends to some extent on DNA sequence. The affinity for specific hetero-ODNs is 50-75-fold higher than the affinity for d(pT)_14-15•d(pA)_14-15. A relative contribution of specific interaction of Fpg with 8-oxoG is comparable at all levels (for minimal ligands, ss and ds ODNs), implying that specific and nonspecific interactions of individual nucleotides of DNA to its total affinity for Fpg is nearly additive. Fpg recognizes free dNMPs through fundamental additive interactions with their structural elements, which are the major contributions of the phosphate moiety. In principle, absolute additivity is not expected for ligands approximating different structural elements of dNMPs. Fpg is able to recognize mononucleotide units within DNA and as free dNMPs in an almost additive manner with comparable affinity. In summary, the interaction of Fpg with specific DNA can be approximated using the model shown in Figure 2.

Figure 2. Thermodynamic model of interactions of Fpg with specific DNA (Zharkov, D.O. et al. 2003)