PDBsum entry 1flz

Hydrolase

PDB id: 1flz

Contents

Protein chain

228 a.a. *

Ligands

URA

Waters ×122

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Stressing-Out DNA? the contribution of serine-Phosphodiester interactions in catalysis by uracil DNA glycosylase.

Authors

R.M.Werner, Y.L.Jiang, R.G.Gordley, G.J.Jagadeesh, J.E.Ladner, G.Xiao, M.Tordova, G.L.Gilliland, J.T.Stivers.

Ref.

Biochemistry, 2000, 39, 12585-12594. [DOI no: 10.1021/bi001532v]

PubMed id

11027138

Abstract

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction. The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Sci. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG.

Secondary reference #1

Title

Crystal structure of escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited.

Authors

G.Xiao, M.Tordova, J.Jagadeesh, A.C.Drohat, J.T.Stivers, G.L.Gilliland.

Ref.

Proteins, 1999, 35, 13-24. [DOI no: 10.1002/(SICI)1097-0134(19990401)35:1<13::AID-PROT2>3.3.CO;2-U]

PubMed id

10090282

Figure 7.
Figure 7. Hypothetical oxycarbenium-ion transition-state for glycosidic bond hydrolysis in DNA.

Figure 8.
Figure 8. Pyrrolidine-based transition-state analog for DNA glycosylases.

The above figures are reproduced from the cited reference with permission from John Wiley & Sons, Inc.