PDBsum entry 1emh

Hydrolase/DNA

PDB id: 1emh

Contents

Protein chain

223 a.a. *

DNA/RNA

Waters ×171

* Residue conservation analysis

PDB id:

1emh

Name:

Hydrolase/DNA

Title:

Crystal structure of human uracil-DNA glycosylase bound to uncleaved substrate-containing DNA

Structure:

DNA (5'-d( Tp Gp Tp (P2u)p Ap Tp Cp Tp T)-3'). Chain: b. Engineered: yes. DNA (5'-d( Ap Ap Ap Gp Ap Tp Ap Ap Cp A)-3'). Chain: c. Engineered: yes. Uracil-DNA glycosylase. Chain: a. Engineered: yes.

Source:

Synthetic: yes. Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Trimer (from PQS)

Resolution:

1.80Å R-factor: 0.216 R-free: 0.233

Authors:

S.S.Parikh,G.Slupphaug,H.E.Krokan,G.M.Blackburn,J.A.Tainer

Key ref:

S.S.Parikh et al. (2000). Uracil-DNA glycosylase-DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupled stereoelectronic effects. Proc Natl Acad Sci U S A, 97, 5083-5088. PubMed id: 10805771 DOI: 10.1073/pnas.97.10.5083

Date:

16-Mar-00 Release date: 16-May-00

Protein chain

P13051  (UNG_HUMAN) -  Uracil-DNA glycosylase from Homo sapiens

Seq: 313 a.a.

Struc: 223 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DNA/RNA chains

T-G-T-P2U-A-T-C-T-T 9 bases

A-A-A-G-A-T-A-A-C-A 10 bases

Enzyme reactions

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

DOI no: 10.1073/pnas.97.10.5083

Proc Natl Acad Sci U S A 97:5083-5088 (2000)

PubMed id: 10805771

Uracil-DNA glycosylase-DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupled stereoelectronic effects.

S.S.Parikh, G.Walcher, G.D.Jones, G.Slupphaug, H.E.Krokan, G.M.Blackburn, J.A.Tainer.

ABSTRACT

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil-DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-A resolution substrate analogue and 2.0-A resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme-DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

Selected figure(s)

Figure 1.
Fig. 1. UDG activity assays for substrate and product DNA constructs. Human UDG cleaves the glycosylic bonds of deoxyuridine and 4'S-dU but not the glycosylic bond of d U (see Methods). This is true even at high concentrations of UDG relative to DNA and over periods of weeks.

Figure 5.
Fig. 5. Structure-based reaction mechanism that resolves the apparent orthogonal paradox for electron transpositions by altering the substrate stereochemistry. (A) A simplified valence-bond representation of the glycosylic bond dissociation hides the paradox that the three electron pairs to be transposed are involved in orthogonal orbitals. (B) In the normal anti-conformation of deoxyuridine, the *-orbital involved in the anomeric effect and the -orbital of the C2==O bond are orthogonal to one another, thus preventing orbital overlap. (C) Severe distortions of the deoxyribose and the glycosylic bond in the strained conformation of deoxyuridine enforced by the UDG active center align the pairs of atomic orbitals participating in each electron transposition, thereby electronically coupling the anomeric and - [Arom] effects to promote bond cleavage.

Figures were selected by an automated process.