Engineering Functional Changes in Escherichia coli Endonuclease III Based on Phylogenetic and Structural Analyses
Autor: | Jeffrey O. Blaisdell, Susan S. Wallace, Jeffrey P. Bond, Takashi Watanabe |
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Rok vydání: | 2005 |
Předmět: |
Molecular Sequence Data
Biology Arginine Biochemistry Catalysis Substrate Specificity Serine Deoxyribonuclease (Pyrimidine Dimer) Structure-Activity Relationship Escherichia coli AP site Lyase activity Molecular Biology Phylogeny Alanine chemistry.chemical_classification Aspartic Acid Base Sequence Escherichia coli Proteins Cell Biology Lyase Protein Structure Tertiary Amino acid chemistry DNA glycosylase Mutagenesis Site-Directed Leucine |
Zdroj: | Journal of Biological Chemistry. 280:34378-34384 |
ISSN: | 0021-9258 |
DOI: | 10.1074/jbc.m504916200 |
Popis: | 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 beta-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. |
Databáze: | OpenAIRE |
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