Uridine nucleosidase (family I)

 

Uracil-DNA glycosylase (UDG) are monofunctional glycosylases and initiate the base excision repair (BER) pathway for uracil by hydrolysing the N-C'1 glycosylic bond between a target uracil and an abasic site. The human BER cycle is important for restoring the chemical integrity of DNA.

The uracil-DNA glycosylase superfamily consists of 6 smaller families, based on sequence alignments. Both human and E.coli UDG come under Family I and are also called UNGs.

The enzyme mechanism has been hotly debated. Classically, an acid/base mechanism has been employed (mechanism proposal 2), but new evidence suggests a steric distortion of the DNA substrate catalyses the reaction in a mechanism similar to SN1 dissociation (mechanism proposal 1).

 

Reference Protein and Structure

Sequence
P12295 UniProt (3.2.2.27) IPR002043 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1eug - CRYSTAL STRUCTURE OF ESCHERICHIA COLI URACIL DNA GLYCOSYLASE AND ITS COMPLEXES WITH URACIL AND GLYCEROL: STRUCTURE AND GLYCOSYLASE MECHANISM REVISITED (1.6 Å) PDBe PDBsum 1eug
Catalytic CATH Domains
3.40.470.10 CATHdb (see all for 1eug)
Click To Show Structure

Enzyme Reaction (EC:3.2.2.3)

water
CHEBI:15377ChEBI
+
2'-deoxyuridine
CHEBI:16450ChEBI
→
uracil
CHEBI:17568ChEBI
+
2-deoxy-D-ribofuranose
CHEBI:90761ChEBI
Alternative enzyme names: Uridine hydrolase, Uridine ribohydrolase,

Enzyme Mechanism

Introduction

This mechanism represents the steric contortion mechanism. Here the tetrahedral distortion is imposed by the structurally rigid walls of the active site, formed by Tyr and Phe residues. The enzyme centre flattens the pucker ring of the uridine deoxyribose, raising the glycosylic bond to a semi-axial position, allowing pi-sigma* overlap. This stereoelectronic effect increases in strength as the substrate is further distorted towards the transition state. As the transition state move towards a tetrahedral geometry, and glycosylic bond rotation occurs, an anomeric effect is coupled to the pi systems of the uracil ring, resulting in orbital overlap of the glycosylic bond and the carbonyl C2 and C4 pi systems. The developing negative charge of the transition state is stabilised by hydrogen bonding to His-187. The enzyme funnels binding energy for use is catalysis by employing substrate distortions to couple two stereoelectronic effects to promote efficient catalysis. The abasic nucleotide relaxes into a more puckered conformation and withdraws from the enzyme while uracil tilts deeper into the active site, improving its stacking interactions with the Phe residue present. These rearrangements lower the product's energies relative to the reactant's, reducing the strain within the active site and allowing the enzyme to bind preferentially to the products.

Catalytic Residues Roles

UniProt PDB* (1eug)
Tyr66 Tyr66A The residue acts to distort the DNA substrate towards the transition state conformation through steric interactions with the pucker ring of the uridine deoxyribose. The residue is also implicated in preventing the binding of thymine within the active site. activator, steric role
Phe77 Phe77A The residue acts to distort the DNA substrate towards the transition state conformation by steric and pi stacking interactions with the pucker ring of the uridine deoxyribose. activator, steric role
His187 His187A The residue hydrogen bonds to the developing anion within the transition state, lowering its energy. hydrogen bond donor, electrostatic stabiliser
Asp64 Asp64A Stabilising and activating the catalytic histidine. May also act as a general acid/base activator, electrostatic stabiliser
*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

intramolecular elimination, intermediate formation, overall reactant used, bimolecular nucleophilic addition, proton transfer, overall product formed, native state of enzyme regenerated, intermediate terminated, tautomerisation (not keto-enol), reaction occurs outside the enzyme

References

  1. Parikh SS et al. (2000), Proc Natl Acad Sci U S A, 97, 5083-5088. Uracil-DNA glycosylase-DNA substrate and product structures: Conformational strain promotes catalytic efficiency by coupled stereoelectronic effects. DOI:10.1073/pnas.97.10.5083. PMID:10805771.
  2. Schormann N et al. (2014), Protein Sci, 23, 1667-1685. Uracil-DNA glycosylases-Structural and functional perspectives on an essential family of DNA repair enzymes. DOI:10.1002/pro.2554. PMID:25252105.
  3. Drohat AC et al. (2014), Org Biomol Chem, 12, 8367-8378. Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA. DOI:10.1039/c4ob01063a. PMID:25181003.
  4. Jiang YL et al. (2003), Biochemistry, 42, 1922-1929. Powering DNA Repair through Substrate Electrostatic Interactions†. DOI:10.1021/bi027014x. PMID:12590578.
  5. Jiang YL et al. (2002), J Biol Chem, 277, 15385-15392. Probing the Limits of Electrostatic Catalysis by Uracil DNA Glycosylase Using Transition State Mimicry and Mutagenesis. DOI:10.1074/jbc.m200634200. PMID:11859082.
  6. Handa P et al. (2002), Nucleic Acids Res, 30, 3086-3095. Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers: evidence for the occurrence of long-range interactions between the enzyme and substrate. PMID:12136091.
  7. Stivers JT et al. (2001), Arch Biochem Biophys, 396, 1-9. Uracil DNA Glycosylase: Insights from a Master Catalyst. DOI:10.1006/abbi.2001.2605. PMID:11716455.
  8. Jiang YL et al. (2001), Biochemistry, 40, 7710-7719. Reconstructing the substrate for uracil DNA glycosylase: tracking the transmission of binding energy in catalysis. PMID:11412125.
  9. Dinner AR et al. (2001), Nature, 413, 752-755. Uracil-DNA glycosylase acts by substrate autocatalysis. DOI:10.1038/35099587. PMID:11607036.
  10. Parikh SS et al. (2000), Mutat Res, 460, 183-199. Lessons learned from structural results on uracil-DNA glycosylase. DOI:10.1016/s0921-8777(00)00026-4. PMID:10946228.
  11. Werner RM et al. (2000), Biochemistry, 39, 14054-14064. Kinetic isotope effect studies of the reaction catalyzed by uracil DNA glycosylase: evidence for an oxocarbenium ion-uracil anion intermediate. PMID:11087352.
  12. Xiao G et al. (1999), Proteins, 35, 13-24. Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited. DOI:10.2210/pdb1eug/pdb. PMID:10090282.
  13. Kavli B et al. (1996), EMBO J, 15, 3442-3447. Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase. PMID:8670846.
  14. Mol CD et al. (1995), Cell, 80, 869-878. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. PMID:7697717.
  15. Savva R et al. (1995), Nature, 373, 487-493. The structural basis of specific base-excision repair by uracil–DNA glycosylase. DOI:10.1038/373487a0. PMID:7845459.

Step 1. The structural constraints imposed upon the uridine deoxyribose ring by Tyr 66 and Phe 77 induce unimolecular elimination to form a transient oxonium species and an anionic precursor of uracil

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Catalytic Residues Roles

Residue Roles
Tyr66A steric role
His187A hydrogen bond donor
Phe77A steric role
Asp64A electrostatic stabiliser

Chemical Components

ingold: intramolecular elimination, intermediate formation, overall reactant used

Step 2. The transient oxonium species is attacked by water, forming the 1-alpha hydroxy group of the D-ribose product. The anionic intermediate is stabilised though hydrogen bonding to His187, and is then protonated from the attacking water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr66A activator, steric role
His187A hydrogen bond donor, electrostatic stabiliser
Phe77A activator, steric role
Asp64A activator

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall product formed, native state of enzyme regenerated, intermediate terminated, intermediate formation

Step 3. The intermediate tautomerises to form the uracil product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

tautomerisation (not keto-enol), reaction occurs outside the enzyme
Download: Image, Marvin File

Step 1. The structural constraints imposed upon the uridine deoxyribose ring by Tyr 66 and Phe 77 induce unimolecular elimination to form a transient oxonium species and an anionic precursor of uracil

Step 2. The transient oxonium species is attacked by water, forming the 1-alpha hydroxy group of the D-ribose product. The anionic intermediate is stabilised though hydrogen bonding to His187, and is then protonated from the attacking water.

Step 3. The intermediate tautomerises to form the uracil product.

Products.

Introduction

This proposal represents the classical acid/base proposal carried out by a cationic His-187 and an absolutely conserved Asp-64. Here the peptide carbonyl and side-chain carboxyl of Asp-64 activate a water molecule that attacks a weakened glycosylic bond. Destabilisation of the N1−C1‘ bond is brought about by distortion or protonation of the uracil O2 by His-187 Nε2.

Catalytic Residues Roles

UniProt PDB* (1eug)
Tyr66 Tyr66A The residue acts to distort the DNA substrate towards the transition state conformation through steric interactions with the pucker ring of the uridine deoxyribose. The residue is also implicated in preventing the binding of thymine within the active site. steric role
Phe77 Phe77A The residue acts to distort the DNA substrate towards the transition state conformation by steric and pi stacking interactions with the pucker ring of the uridine deoxyribose. steric role
His187 His187A The residue hydrogen bonds to the developing anion within the transition state, lowering its energy. hydrogen bond donor, electrostatic stabiliser
Asp64 Asp64A Acts as a general acid/base. proton acceptor, electrostatic stabiliser, 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, bimolecular nucleophilic substitution, overall product formed, proton transfer, assisted tautomerisation (not keto-enol), native state of enzyme regenerated

References

  1. Shroyer MJ et al. (1999), Biochemistry, 38, 4834-4845. Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis. DOI:10.1021/bi982986j. PMID:10200172.

Step 1. Asp64 abstracts a proton from the catalytic water molecule. The activated hydroxide then attacks the anomeric carbon in a nucleophilic substitution reaction.

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Catalytic Residues Roles

Residue Roles
Asp64A electrostatic stabiliser
Tyr66A steric role
His187A hydrogen bond donor
Phe77A steric role
Asp64A proton acceptor

Chemical Components

intermediate formation, overall reactant used, ingold: bimolecular nucleophilic substitution, overall product formed

Step 2. The intermediate abstracts a proton from Asp64 to regenerate the active site and form the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr66A steric role
Phe77A steric role
His187A electrostatic stabiliser
Asp64A proton donor

Chemical Components

proton transfer, overall product formed, assisted tautomerisation (not keto-enol), native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp64 abstracts a proton from the catalytic water molecule. The activated hydroxide then attacks the anomeric carbon in a nucleophilic substitution reaction.

Step 2. The intermediate abstracts a proton from Asp64 to regenerate the active site and form the final product.

Products.

Introduction

The imidazole group of His-187 catalyses a direct nucleophilic attack on the N1−C1‘ glycosylic bond of uracil and a second nucleophilic attack of a water molecule provides H- and OH-group addition to the N1 and C1‘ atoms, respectively.

Catalytic Residues Roles

UniProt PDB* (1eug)
Tyr66 Tyr66A The residue acts to distort the DNA substrate towards the transition state conformation through steric interactions with the pucker ring of the uridine deoxyribose. The residue is also implicated in preventing the binding of thymine within the active site steric role
Phe77 Phe77A The residue acts to distort the DNA substrate towards the transition state conformation by steric and pi stacking interactions with the pucker ring of the uridine deoxyribose. steric role
His187 His187A Acts as a nucleophile and forms a covalent intermediate with the sugar ring of DNA. covalently attached, nucleofuge, nucleophile
Asp64 Asp64A Stabilising and activating the catalytic histidine and water molecule. activator, electrostatic stabiliser, increase acidity
*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

bimolecular nucleophilic substitution, overall reactant used, intermediate formation, enzyme-substrate complex formation, proton transfer, overall product formed, native state of enzyme regenerated, enzyme-substrate complex cleavage

References

  1. Mol CD et al. (1995), Cell, 80, 869-878. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. PMID:7697717.

Step 1. His187 initiates a nucleophilic attack on the anomeric carbon of the DNA base.

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Catalytic Residues Roles

Residue Roles
Phe77A steric role
Tyr66A steric role
Asp64A electrostatic stabiliser
His187A nucleophile

Chemical Components

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

Step 2. The uracil undergoes tautomerisation and abstracts a proton from the catalytic water molecule, forming one of the final products and activating the water.

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Catalytic Residues Roles

Residue Roles
Asp64A increase acidity
Tyr66A steric role
Phe77A steric role
His187A covalently attached

Chemical Components

proton transfer, overall reactant used, overall product formed

Step 3. The activated water then attacks the anomeric carbon of the enzyme bound sugar. This forms the final product and regenerates the active site.

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Catalytic Residues Roles

Residue Roles
Asp64A activator
Tyr66A steric role
Phe77A steric role
His187A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, native state of enzyme regenerated, overall product formed, enzyme-substrate complex cleavage
Download: Image, Marvin File

Step 1. His187 initiates a nucleophilic attack on the anomeric carbon of the DNA base.

Step 2. The uracil undergoes tautomerisation and abstracts a proton from the catalytic water molecule, forming one of the final products and activating the water.

Step 3. The activated water then attacks the anomeric carbon of the enzyme bound sugar. This forms the final product and regenerates the active site.

Products.

Introduction

This proposal represents the second acid/base proposal carried out by a neutral His-187 and an absolutely conserved Asp-64. Here His-187 activates a water molecule that attacks a weakened glycosylic bond.

Catalytic Residues Roles

UniProt PDB* (1eug)
Tyr66 Tyr66A The residue acts to distort the DNA substrate towards the transition state conformation through steric interactions with the pucker ring of the uridine deoxyribose. The residue is also implicated in preventing the binding of thymine within the active site. steric role
Phe77 Phe77A The residue acts to distort the DNA substrate towards the transition state conformation by steric and pi stacking interactions with the pucker ring of the uridine deoxyribose. steric role
His187 His187A Acts as a general acid/base. proton acceptor, proton donor
Asp64 Asp64A Activates the catalytic water molecule. electrostatic stabiliser, increase acidity
*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

overall product formed, bimolecular nucleophilic substitution, overall reactant used, intermediate formation, native state of enzyme regenerated, assisted tautomerisation (not keto-enol), proton transfer

References

  1. Drohat AC et al. (2014), Org Biomol Chem, 12, 8367-8378. Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA. DOI:10.1039/c4ob01063a. PMID:25181003.

Step 1. His-187 abstracts a proton from the catalytic water molecule. The activated hydroxide then attacks the anomeric carbon in a nucleophilic substitution reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe77A steric role
Tyr66A steric role
Asp64A electrostatic stabiliser
Asp64A increase acidity
His187A proton acceptor

Chemical Components

overall product formed, ingold: bimolecular nucleophilic substitution, overall reactant used, intermediate formation

Step 2. The intermediate abstracts a proton from His-187 to regenerate the active site and form the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe77A steric role
Tyr66A steric role
His187A proton donor

Chemical Components

native state of enzyme regenerated, assisted tautomerisation (not keto-enol), overall product formed, proton transfer
Download: Image, Marvin File

Step 1. His-187 abstracts a proton from the catalytic water molecule. The activated hydroxide then attacks the anomeric carbon in a nucleophilic substitution reaction.

Step 2. The intermediate abstracts a proton from His-187 to regenerate the active site and form the final product.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, James W. Murray, Craig Porter, Morwenna Hall