Structural and Biochemical Exploration of a Critical Amino Acid in Human 8-Oxoguanine Glycosylase
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- 作者：Derek P. G. Norman ; Sang J. Chung ; and Gregory L. Verdine
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：February 18, 2003
- 年：2003
- 卷：42
- 期：6
- 页码：1564 - 1572
- 全文大小：351K
- 年卷期：v.42,no.6(February 18, 2003)
- ISSN：1520-4995
文摘
Members of the HhH-GPD superfamily of DNA glycosylases are responsible for the recognitionand removal of damaged nucleobases from DNA. The hallmark of these proteins is a motif comprisinga helix-hairpin-helix followed by a Gly/Pro-rich loop and terminating in an invariant, catalytically essentialaspartic acid residue. In this study, we have probed the role of this Asp in human 8-oxoguanine DNAglycosylase (hOgg1) by mutating it to Asn (D268N), Glu (D268E), and Gln (D268Q). We show that thisaspartate plays a dual role, acting both as an N-terminal 
-helix cap and as a critical residue for catalysisof both base excision and DNA strand cleavage by hOgg1. Mutation of this residue to asparagine, anotherhelix-capping residue, preserves stability of the protein while drastically reducing enzymatic activity. Acrystal structure of this mutant is the first to reveal the active site nucleophile Lys249 in the presence oflesion-containing DNA; this structure offers a tantalizing suggestion that base excision may occur bycleavage of the glycosidic bond and then attachment of Lys249. Mutation of the aspartic acid to glutamineand glutamic acid destabilizes the protein fold to a significant extent but, surprisingly, preserves catalyticactivity. Crystal structures of these mutants complexed with an unreactive abasic site in DNA revealthese residues to adopt a sterically disfavored helix-capping conformation.
-helix cap and as a critical residue for catalysisof both base excision and DNA strand cleavage by hOgg1. Mutation of this residue to asparagine, anotherhelix-capping residue, preserves stability of the protein while drastically reducing enzymatic activity. Acrystal structure of this mutant is the first to reveal the active site nucleophile Lys249 in the presence oflesion-containing DNA; this structure offers a tantalizing suggestion that base excision may occur bycleavage of the glycosidic bond and then attachment of Lys249. Mutation of the aspartic acid to glutamineand glutamic acid destabilizes the protein fold to a significant extent but, surprisingly, preserves catalyticactivity. Crystal structures of these mutants complexed with an unreactive abasic site in DNA revealthese residues to adopt a sterically disfavored helix-capping conformation.