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. 2000 May 9;97(10):5083-8.
doi: 10.1073/pnas.97.10.5083.

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

S S Parikh  1 , G Walcher, G D Jones, G Slupphaug, H E Krokan, G M Blackburn, J A Tainer
Affiliations

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

S S Parikh et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 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 dΨU (see Methods). This is true even at high concentrations of UDG relative to DNA and over periods of weeks.
Figure 2
Figure 2
Cocrystal structures of UDG bound to uncleaved substrate and cleaved product DNA. (A) dΨU-containing DNA (orange) binds UDG near the C-terminal end of its central β-sheet (dark blue arrows), which is surrounded by eight α-helices (purple). (B) Experimental electron density defines the stereochemical deformation and intact bond for the substrate dΨU complex. The glycosylic bond of the dΨU (orange carbon tubes, red oxygens, blue nitrogens, yellow phosphorus) is not cleaved, as demonstrated by the simulated-annealed omit map (blue) contoured at 2σ. The normally trigonal planar 1-position is clearly distorted out of the plane of the uracil ring toward a tetrahedral geometry. Difference maps of the active-site center are flat, indicating that the distortion is accurately depicted by the crystal structure.
Figure 3
Figure 3
A UDG global conformational change on binding substrate DNA creates the enzyme active center, which thereafter remains unchanged during the glycosylic bond cleavage reaction. (A) Superposition of apo-UDG (green) and DNA-bound UDG (dark blue) with the uncleaved substrate DNA (orange) shows that UDG undergoes an architecturally determined conformational closing on binding substrate DNA. β1 and β3 increase the number of interstrand hydrogen bonds between them and thereby zip up the UDG β-zipper. This creates the catalytically competent active site by bringing L272 into the DNA base stack and H268 and D145 into the active center. (B) Superposition of the uncleaved-substrate (orange) and product (pink) UDG–DNA complexes reveals the basis of glycosylic bond cleavage by UDG. As the conformation of UDG (dark blue carbon tubes) remains unchanged throughout the reaction (see text), only the protein conformation from the dΨU structure is shown.
Figure 4
Figure 4
Deoxyuridine (gray carbon tubes, red oxygens, blue nitrogens) in DNA is severely distorted by the UDG active center to achieve the observed conformation of the dΨU (orange carbon tubes, red oxygens, blue nitrogens). The left side of the large arrow is deoxyuridine in the conformation normally found in DNA. The arrow implies the observed flipping of the substrate nucleotide out of the DNA helix, which results in the altered position of the 5′P. When flipped into the UDG active center (right side of large arrow), the uracil ring is rotated ≈90° on its N1–C4 axis to a χ angle of 177°. Furthermore, the deoxyribose sugar of the enzyme bound substrate is flattened to a mild C3′-exo, which raises the uracil to a semiaxial position. The normally trigonal planar 1-position of uracil is strained to an almost tetrahedral geometry. The small arrows indicate the steric hindrance, which causes the deformation at the uracil 1-position. The conformation of deoxyuridine in DNA (gray) is derived from the conformation of deoxythymidine in a G/T mismatch (Protein Data Bank accession code 113D).
Figure 5
Figure 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 σ*-orbital involved in the anomeric effect and the π-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 σ-πArom effects to promote bond cleavage.
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