DNA-formamidopyrimidine glycosylase

 

Formamidopyrimidine-DNA glycosylase, otherwise known as Fpg or MutM, is a bifunctional base excision repair enzyme - it has both DNA glycosylase and AP lyase activities and is involved in removal of oxidised bases including 2,6-diamino-4-hydroxy- 5-formamidopyrimidine (FapydG) and 8-oxoguanine from oxidatively-damaged DNA in initiation of the base excision repair pathway.

 

Reference Protein and Structure

Sequence
P05523 UniProt (3.2.2.23, 4.2.99.18) IPR020629 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1k82 - Crystal structure of E.coli formamidopyrimidine-DNA glycosylase (Fpg) covalently trapped with DNA (2.1 Å) PDBe PDBsum 1k82
Catalytic CATH Domains
3.20.190.10 CATHdb (see all for 1k82)
Click To Show Structure

Enzyme Reaction (EC:3.2.2.23)

a ring-opened 7-methylguanine in DNA
CHEBI:137936ChEBI
→
2,6-diamino-4-hydroxy-5-(N-methylformamido)pyrimidine
CHEBI:28643ChEBI
+
DNA without the base
CHEBI:137219ChEBI
Alternative enzyme names: 2,6-diamino-4-hydroxy-5(N-methyl)formamidopyrimidine-DNA glycosylase, 2,6-diamino-4-hydroxy-5N-formamidopyrimidine-DNA glycosylase, Fapy-DNA glycosylase, Fpg protein, Deoxyribonucleate glycosidase, Formamidopyrimidine-DNA glycosylase,

Enzyme Mechanism

Introduction

Bifunctional DNA glycosylases differ from monofunctional glycosylases in that they form a characteristic covalent Schiff base-like intermediate. Deoxyribose sugar protonation by Glu2 enables Pro1 to nucleophilically attack the oxidised base, opening up the ribose ring. The resulting intermediate undergoes deprotonation twice and subsequent reprotonation which facilitates the cleavage of the N-glycosydic bond, excising the oxidised base. The resultant Schiff base-like species then enters the enzyme's AP lyase catalytic mechanism where it undergoes beta and delta elimination. The mechanism described here is base independent, where both 8-oxoguanine and FapydG have been shown to excise via the same mechanism. Other residues of the enzyme will determine the exact specificity prior to catalytic initiation. For this reason, between the two proposed mechanisms this is favoured as it supports the idea Fpg has a wide substrate specificity.

Catalytic Residues Roles

UniProt PDB* (1k82)
Pro2 (N-term) Pro1A(I) (N-term) Nucleophilically attacks the deoxyribose ring to cause ring cleavage and subsequently is deprotonated by Glu2 to form a Schiff base intermediate. electron pair donor, covalently attached, nucleophile, proton donor
Glu3 Glu2A(I) Initiates the catalytic mechanism by protonating the ribose sugar and involved in proton transfer reactions later on to cleave the N-glycosidic bond. proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, overall reactant used, intermediate formation, enzyme-substrate complex formation, bimolecular nucleophilic substitution, intermediate collapse, overall product formed, elimination (not covered by the Ingold mechanisms)

References

  1. Blank ID et al. (2015), Sci Rep, 5, 10369-. A base-independent repair mechanism for DNA glycosylase--no discrimination within the active site. DOI:10.1038/srep10369. PMID:26013033.
  2. Dizdaroglu M et al. (2017), Mutat Res Rev Mutat Res, 771, 99-127. Repair of oxidatively induced DNA damage by DNA glycosylases: Mechanisms of action, substrate specificities and excision kinetics. DOI:/10.1016/j.mrrev.2017.02.001.
  3. Sadeghian K et al. (2014), Angew Chem Int Ed Engl, 53, 10044-10048. Ribose-protonated DNA base excision repair: a combined theoretical and experimental study. DOI:10.1002/anie.201403334. PMID:25065673.
  4. Serre L et al. (2002), EMBO J, 21, 2854-2865. Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA. DOI:10.1093/emboj/cdf304. PMID:12065399.
  5. Gilboa R et al. (2002), J Biol Chem, 277, 19811-19816. Structure of Formamidopyrimidine-DNA Glycosylase Covalently Complexed to DNA. DOI:10.1074/jbc.m202058200. PMID:11912217.

Step 1. Glu2 protonates the deoxyribose ring, facilitating nucleophilic attack by Pro1 and the opening of the deoxyribose ring.

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Catalytic Residues Roles

Residue Roles
Pro1A(I) (N-term) covalently attached, nucleophile
Glu2A(I) proton donor

Chemical Components

proton transfer, overall reactant used, intermediate formation, enzyme-substrate complex formation, ingold: bimolecular nucleophilic substitution

Step 2. Glu2 reorientates itself and deprotonates Pro1.

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Catalytic Residues Roles

Residue Roles
Pro1A(I) (N-term) covalently attached
Glu2A(I) proton acceptor
Pro1A(I) (N-term) proton donor

Chemical Components

proton transfer, intermediate collapse

Step 3. The nitrogen of the oxidised base is protonated by the hydroxyl group on the open ring deoxyribose which itself then abstracts a proton from Glu2. This facilitates N-glycosydic bond cleavage resulting in the formation of an excised base and Schiff base- like covalent intermediate.

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Catalytic Residues Roles

Residue Roles
Pro1A(I) (N-term) covalently attached
Glu2A(I) proton donor
Pro1A(I) (N-term) electron pair donor

Chemical Components

proton transfer, overall product formed, elimination (not covered by the Ingold mechanisms), intermediate collapse
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Step 1. Glu2 protonates the deoxyribose ring, facilitating nucleophilic attack by Pro1 and the opening of the deoxyribose ring.

Step 2. Glu2 reorientates itself and deprotonates Pro1.

Step 3. The nitrogen of the oxidised base is protonated by the hydroxyl group on the open ring deoxyribose which itself then abstracts a proton from Glu2. This facilitates N-glycosydic bond cleavage resulting in the formation of an excised base and Schiff base- like covalent intermediate.

Products.

Introduction

Deoxyribose ring protonation and subsequent nucleophilic attack of Pro1 results in the opening of the deoxyribose ring. Multiple protonation events between the residues, substrate and a water molecule cause the cleavage of the N-glycosidic bond between the sugar and base to leave a Schiff base-like intermediate and 8-oxoguanine. The mechanism described here is specific to 8-oxoguanine, unlike the alternative mechanism proposal where 8-oxoguanine can be excised in a similar fashion to FapydG.

Catalytic Residues Roles

UniProt PDB* (1k82)
Pro2 (N-term) Pro1A(I) (N-term) Nucleophilically attacks the deoxyribose ring to cause ring cleavage and subsequently is deprotonated by a hydroxide group (originally water that has been deprotonated by 8-oxoguanine) to form a covalent Schiff base-like intermediate. electron pair donor, covalently attached, nucleophile, proton donor
Glu3 Glu2A(I) Initiates the catalytic mechanism by protonating the ribose sugar and involved in proton transfer reactions later on resulting in cleavage of the N-glycosidic bond. proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

intermediate formation, overall reactant used, proton transfer, bimolecular nucleophilic substitution, enzyme-substrate complex formation, proton relay, intermediate collapse, heterolysis, elimination (not covered by the Ingold mechanisms), overall product formed

References

  1. Kreppel A et al. (2018), J Am Chem Soc, 140, 4522-4526. Base-Independent DNA Base-Excision Repair of 8-Oxoguanine. DOI:10.1021/jacs.7b11254. PMID:29578340.
  2. Sadeghian K et al. (2014), Angew Chem Int Ed Engl, 53, 10044-10048. Ribose-protonated DNA base excision repair: a combined theoretical and experimental study. DOI:10.1002/anie.201403334. PMID:25065673.
  3. Serre L et al. (2002), EMBO J, 21, 2854-2865. Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA. DOI:10.1093/emboj/cdf304. PMID:12065399.

Step 1. Glu2 protonates the ribose ring and Pro1 nucleophilically attacks C1, opening up the deoxyribose ring.

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Catalytic Residues Roles

Residue Roles
Pro1A(I) (N-term) nucleophile
Glu2A(I) proton donor

Chemical Components

intermediate formation, overall reactant used, proton transfer, ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation

Step 2. Proton relay from the Pro1 to the 8-oxoguanine in the same intermediate via a water molecule.

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Catalytic Residues Roles

Residue Roles
Pro1A(I) (N-term) covalently attached, proton donor

Chemical Components

proton relay, intermediate collapse

Step 3. Proton relay within the intermediate resulting in the protonation of Glu2.

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Catalytic Residues Roles

Residue Roles
Pro1A(I) (N-term) covalently attached
Glu2A(I) proton acceptor

Chemical Components

proton transfer, proton relay, intermediate collapse

Step 4. Protonation of N in 8-oxoguanine by the open ribose ring results in the cleavage of the N-glycosidic bond. The deprotonated open deoxyribose can abstract a proton from Glu2. This results in the formation of an excised oxidised base and a Schiff base-like intermediate which is then subject to AP lyase enzymatic activity.

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Catalytic Residues Roles

Residue Roles
Glu2A(I) proton donor
Pro1A(I) (N-term) electron pair donor

Chemical Components

heterolysis, elimination (not covered by the Ingold mechanisms), proton relay, proton transfer, overall product formed, intermediate collapse
Download: Image, Marvin File

Step 1. Glu2 protonates the ribose ring and Pro1 nucleophilically attacks C1, opening up the deoxyribose ring.

Step 2. Proton relay from the Pro1 to the 8-oxoguanine in the same intermediate via a water molecule.

Step 3. Proton relay within the intermediate resulting in the protonation of Glu2.

Step 4. Protonation of N in 8-oxoguanine by the open ribose ring results in the cleavage of the N-glycosidic bond. The deprotonated open deoxyribose can abstract a proton from Glu2. This results in the formation of an excised oxidised base and a Schiff base-like intermediate which is then subject to AP lyase enzymatic activity.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, Morwenna Hall