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Literature summary for 5.6.2.1 extracted from

  • Strahs, D.; Zhu, C.X.; Cheng, B.; Chen, J.; Tse-Dinh, Y.C.
    Experimental and computational investigations of Ser10 and Lys13 in the binding and cleavage of DNA substrates by Escherichia coli DNA topoisomerase I (2006), Nucleic Acids Res., 34, 1785-1797.
    View publication on PubMedView publication on EuropePMC
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Cloned(Commentary)

Cloned (Comment) Organism
-
 Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
-
 Escherichia coli

Protein Variants

Protein Variants Comment Organism
K13A Investigation of the potential catalytic role of the serine and lysine residue conserved in the active site region of bacterial topoisomerase I Ser10 and Lys13 of Escherichia coli DNA topoisomerase I are mutated to alanines. 50-to 100fold reduction in relaxation activity, not affected in non-covalent DNA binding, mutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Ala13 is unable to make interactions with the acidic residues of the active site. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, rates of cleavage are too low to be analyzed Escherichia coli
K13R Arg13 residue is a larger residue than lysine, and is unable to fit in the space assigned to Lys13. Reduced DNA binding affinity, possibly changing Ser10 to a threonine residue, and changing Lys13 to arginine allow retention of activity, relaxation activity assays show that introduction of a different side chain with similar functionality but different steric properties at these two positions result in even lower relaxation activitymutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Do not have severe changes in protein folding. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, rates of cleavage are too low to be analyzed Escherichia coli
S10A Investigation of the potential catalytic role of the serine and lysine residue conserved in the active site region of bacterial topoisomerase I Ser10 and Lys13 of Escherichia coli DNA topoisomerase I are mutated to alanines. 5-to 10fold reduction in relaxation activity, not affected in non-covalent DNA binding, mutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Ala10 is unable to form a hydrogen bond with phosphate 8. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, yield of the cleaved complex observed is 30fold lower than the wild-type enzyme at each enzyme: substrate ratio even after 60 min of incubation Escherichia coli
S10T Reduced DNA binding affinity, possibly changing Ser10 to a threonine residue, and changing Lys13 to arginine allow retention of activity, relaxation activity assays show that introduction of a different side chain with similar functionality but different steric properties at these two positions result in even lower relaxation activitymutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Thr10 forms a hydrogen bond with phosphate 8. Do not have severe changes in protein folding. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, rates of cleavage are too low to be analyzed Escherichia coli

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
97000
-
 SDS–PAGE Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) Organism
by combination of phosphocellulose, hydroxylapitite and ssDNA agarose column chromatography as described elsewhere Escherichia coli

Synonyms

Synonyms Comment Organism
DNA topoisomerase I
-
 Escherichia coli
Topoisomerase I
-
 Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.5
-
 While wild-type enzyme is active at pH 5.5, S10A mutant requires pH to be elevated to 6.0 for relaxation activity to be observed, suggesting a protonation event between pH 5.5–6.0 in the S10A mutant to influence the activity Escherichia coli