PDBsum entry 2c56

Hydrolase

PDB id: 2c56

Contents

Protein chain

228 a.a. *

Ligands

GLC-FRU

Waters ×259

* Residue conservation analysis

PDB id:

2c56

Name:

Hydrolase

Title:

A comparative study of uracil DNA glycosylases from human and herpes simplex virus type 1

Structure:

Uracil DNA glycosylase. Chain: a. Engineered: yes. Mutation: yes

Source:

Human herpesvirus 1. Organism_taxid: 10298. Expressed in: escherichia coli. Expression_system_taxid: 511693.

Resolution:

2.10Å R-factor: 0.151 R-free: 0.212

Authors:

K.Krusong,E.P.Carpenter,S.R.W.Bellamy,R.Savva,G.S.Baldwin

Key ref:

K.Krusong et al. (2006). A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1. J Biol Chem, 281, 4983-4992. PubMed id: 16306042 DOI: 10.1074/jbc.M509137200

Date:

26-Oct-05 Release date: 28-Nov-05

Protein chain

P10186  (UNG_HHV11) -  Uracil-DNA glycosylase from Human herpesvirus 1 (strain 17)

Seq: 334 a.a.

Struc: 228 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.2.27 - uracil-DNA glycosylase.

DOI no: 10.1074/jbc.M509137200

J Biol Chem 281:4983-4992 (2006)

PubMed id: 16306042

A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1.

K.Krusong, E.P.Carpenter, S.R.Bellamy, R.Savva, G.S.Baldwin.

ABSTRACT

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.

Selected figure(s)

Figure 5.
FIGURE 5. Binding of the human wild-type and D145N/H268N UNG enzymes to non-cleavable substrate analogs d rd and -FdUrd. The binding of hexachlorofluorescein-labeled oligonucleotides containing the non-cleavable substrate analogs d rd and -FdUrd was monitored using fluorescence polarization. A, the binding of wild-type hUNG was measured with d rd (•) and -FdUrd ( ). Data are shown with the best fit to the binding equation with the following values: d rd, K[d] = 4.4 ± 0.5 µM, A[D] = 0.041 ± 0.002, and A[D][E] = 0.17 ± 0.003; and -FdUrd, K[d] = 6.3 ± 0.6 µM, A[D] = 0.039 ± 0.002, and A[D][E] = 0.13 ± 0.002. B, the binding of human D145N/H268N UNG was measured with d rd (•) and -FdUrd ( ). Data are shown with the best fit to the binding equation with the following values: d rd, K[d] = 3.2 ± 0.2 µM, A[D] = 0.038 ± 0.001, and A[D][E] = 0.14 ± 0.001; and -FdUrd, K[d] = 2.2 ± 0.2 µM, A[D] = 0.041 ± 0.003, and A[D][E] = 0.17 ± 0.002.

Figure 6.
FIGURE 6. Active site of HSV-1 D88N/H210N UNG. A, the amino acid residues in the active site of HSV-1 D88N/H210N UNG (green, carbons; blue, nitrogen; red, oxygens) are aligned with those of the wild-type enzyme (purple). Electron density for HSV-1 D88N/H210N UNG is shown. B, HSV-1 D88N/H210N UNG with uridine bound in the active site is aligned with the wild-type enzyme with uracil bound in the active site (purple). Electron density for HSV-1 D88N/H210N UNG is shown. C, hydrogen bond distances between the residues in the HSV-1 D88N/H210N UNG active site and the bound uracil are shown in red, whereas those in the active site of the wild-type enzyme are shown in black.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 4983-4992) copyright 2006.

Figures were selected by an automated process.