Engineering Functional Changes in Escherichia coli Endonuclease III Based on Phylogenetic and Structural Analyses

Escherichia coli endonuclease III (EcoNth) plays an important cellular role by removing premutagenic pyrimidine damages produced by reactive oxygen species. EcoNth is a bifunctional enzyme that has DNA glycosylase and apurinic/apyrimidinic lyase activities. Using a phylogeny of natural sequences, we...

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Bibliographic Details
Published in:The Journal of biological chemistry 2005-10, Vol.280 (40), p.34378-34384
Main Authors: Watanabe, Takashi, Blaisdell, Jeffrey O., Wallace, Susan S., Bond, Jeffrey P.
Format: Article
Language:English
Subjects:
Arginine
Aspartic Acid
Base Sequence
Catalysis
Deoxyribonuclease (Pyrimidine Dimer) - genetics
Deoxyribonuclease (Pyrimidine Dimer) - metabolism
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Phylogeny
Protein Structure, Tertiary
Serine
Structure-Activity Relationship
Substrate Specificity
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Summary:Escherichia coli endonuclease III (EcoNth) plays an important cellular role by removing premutagenic pyrimidine damages produced by reactive oxygen species. EcoNth is a bifunctional enzyme that has DNA glycosylase and apurinic/apyrimidinic lyase activities. Using a phylogeny of natural sequences, we selected to study EcoNth serine 39, aspartate 44, and arginine 184, which are presumed to be in the vicinity of the damaged base in the glycosylase-substrate complex. These three amino acids are highly conserved among Nth orthologs, although not among homologous glycosylases, such as MutY, that have different base specificities and no lyase activity. To examine the role of these amino acids in catalysis, we constructed three mutants of EcoNth, in which Ser39 was replaced with leucine (S39L), Asp44 was replaced with valine (D44V), and Arg184 was replaced with alanine (R184A), which are the corresponding residues in EcoMutY. We showed that EcoNth S39L does not have significant glycosylase activity for oxidized pyrimidines, although it maintained AP lyase activity. In contrast, EcoNth D44V retained glycosylase activity against oxidized pyrimidines, but the apparent rate constant for the lyase activity of EcoNth D44V was significantly lower than that of EcoNth, indicating that Asp44 in EcoNth is required for β-elimination. Finally, EcoNth R184A maintained lyase activity but exhibited glycosylase specificity different from that of EcoNth. The functional consequences of each of these three substitutions can be rationalized in the context of high resolution protein structures. Thus phylogeny-based scanning mutagenesis has allowed us to identify novel roles for amino acids in the substrate binding pocket of EcoNth in base recognition and/or catalysis.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M504916200