Abstract
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.
Highlights
Ety of damaged pyrimidines and formamidopyrimidines and is a bifunctional enzyme that contains both glycosylase and AP lyase activities [1,2,3]
Denaturing gel analysis of the cleaved products produced by EcoNth R184A on a substrate containing DHU opposite G showed that the glycosylase/lyase activity (Fig. 2A, lane 20) was similar to that of EcoNth
Monofunctional Glycosylase Activity—The absence of substrate cleavage by EcoNth Ser39 was replaced with leucine (S39L) or D44V is consistent with loss of glycosylase activity, loss of lyase activity, or loss of both activities
Summary
Ety of damaged pyrimidines and formamidopyrimidines and is a bifunctional enzyme that contains both glycosylase and AP lyase activities [1,2,3]. Nth is conserved over all three phylogenetic domains, and with respect to qualitative functional features (that is, glycosylase and lyase activity), EcoNth represents the activity of orthologous proteins and their common ancestor. When the crystal structure of the archetypical member of the Nth family, EcoNth, was solved [25, 26], a number of motifs were identified that are shared by relatives of EcoNth. The helix-hairpin-helix (HhH) motif, followed by a Gly/Pro-rich loop and a conserved aspartic acid (HhH-GPD motif), and the iron sulfur cluster loop, are conserved in almost all of the Nth orthologs [26]. EcoMutY contains both the HhH motif and the iron sulfur cluster [24, 27] It appears that Nth and MutY were derived from a common ancestral gene. EcoNth R184A exhibited a qualitatively different substrate specificity from that of EcoNth
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