PDBsum entry 1emh

Hydrolase/DNA

PDB id: 1emh

Contents

Protein chain

223 a.a. *

DNA/RNA

Waters ×171

* Residue conservation analysis

References listed in PDB file

Key reference

Title

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

Authors

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

Ref.

Proc Natl Acad Sci U S A, 2000, 97, 5083-5088. [DOI no: 10.1073/pnas.97.10.5083]

PubMed id

10805771

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.

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.

Secondary reference #1

Title

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-Dna glycosylase with DNA.

Authors

S.S.Parikh, C.D.Mol, G.Slupphaug, S.Bharati, H.E.Krokan, J.A.Tainer.

Ref.

EMBO J, 1998, 17, 5214-5226. [DOI no: 10.1093/emboj/17.17.5214]

PubMed id

9724657

Figure 3.
Figure 3 Stereo views of the electron density and atomic coordinates showing wtUDG and L272A recognition of uracil-containing and AP site DNA. The bias-reduced, [A]-weighted 2F[obs]-F[calc] electron density map, contoured at 2 (blue) and 5 (pink), is shown for the complexes (see text). (A) The wtUDG/U A DNA complex. The electron density for the entire length of the 45° bent DNA is shown, with protein and DNA atoms as yellow (carbon), red (oxygen), blue (nitrogen) and green (phosphorus) tubes. Pro271, Leu272 and Ser273 penetrate the DNA minor groove, with the Leu272 side chain inserted into the DNA base stack opposite the uracil orphan base partner, adenine. (B) The wtUDG/U

Figure 5.
Figure 5 Initial damage detection by UDG Ser -Pro pinch. (A) Backbone compression forced by three Ser -Pro-rich loops. The loops of the Ser -Pro pinch (green) compress the uracil-containing DNA strand at the phosphates 5' and 3' of the uracil nucleotide in the directions indicated by the arrows. The wtUDG/U A structure is shown, but this compression occurs in all of the complex structures. (B) The UDG Ser -Pro pinch for initial damage detection. The initial UDG -DNA complex is recreated by superimposing straight B-DNA (white) onto the kinked DNA seen in the co-crystal structures (orange), and the structure of uncomplexed wtUDG (stippled magenta ribbons) onto the DNA-bound enzyme. The view is looking into the DNA major groove at the Leu272 loop (center) and the 4-Pro loop (165 -169). These Ser -Pro loops, along with the Gly -Ser loop (246 -247), interact with the DNA phosphodiester backbone, compressing the intrastrand phosphate distance of the uracil-containing DNA strand and kinking DNA.

The above figures are reproduced from the cited reference which is an Open Access publication published by Macmillan Publishers Ltd