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Catalytic Site Atlas

CSA LITERATURE entry for 1ecl

E.C. nameDNA topoisomerase
SpeciesEscherichia coli (Bacteria)
E.C. Number (IntEnz)
CSA Homologues of 1eclThere are 20 Homologs
CSA Entries With UniProtID P06612
CSA Entries With EC Number
PDBe Entry 1ecl
PDBSum Entry 1ecl
MACiE Entry 1ecl

Literature Report

IntroductionType I DNA topoisomerase enzymes alter the topology of DNA by transiently breaking one or two strands of DNA, passing a single or double strand through the break and then resealing the break. This allows for the interconversion of topological isomers, which is necessary for a number of cellular transactions such as replication, transcription and recombination. The enzyme is a member of the IA sub family, classified by the covalently bound enzyme-DNA intermediate.
MechansimThe enzyme mechanism is generally accepted to proceed via two sequential trans-esterification reactions. Initially, the enzyme recognises and binds a DNA region, positioning the DNA in the active site. Cleavage of the single stranded DNA occurs via the nucleophilic attack of the Tyr 319 hydroxyl group at the 5' phosphoryl end, with the formation of a covalent bond between the two, while the 3' end remains non-covalently bound to the enzyme. The enzyme opens to allow the passage of the other strand through the gap created by separating the broken ends of the DNA. The gap closes, trapping the DNA strand, and acts to re-ligate the cleaved strand. The enzyme then opens to release both the re-ligated strand and the strand held in the transient gap.
It should be noted that the invariant His 365 has not been identified as catalytic on the experimental evidence given in PMID: 11809772, contradicting earlier references.

Catalytic Sites for 1ecl

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
TyrA319319macie:sideChainThe OH group of the residue acts as a nucleophile towards an internucleoside phosphate group, resulting in a covalently bound enzyme-DNA intermediate. It has been suggested that the residue is deprotonated before nucleophilic attack occurs, although no general base has been identified. The proton is transferred to the deoxyribose 3' oxygen of the DNA substrate from the scissile phosphate.
The residue interacts with the positively charged side chain of Arg 321, modifying its associated pKa and stabilising the charged phenolate intermediate.
GluA99macie:sideChainThe residue acts as a general base in proton removal from the 3' hydroxyl group during DNA rejoining, and a proton donor during DNA breakage. The large distance between the residue and the catalytic Tyr 319 suggest that it does not act as a direct base to the nucleophile.
ArgA321321macie:sideChainThe positively charged side chain interacts with the oxygen of scissile phosphate group, and is implicated in the modification of the Tyr 319 pKa by stabilising the charged phenolate intermediate.

Literature References

Chen SJ
Identification of active site residues in Escherichia coli DNA topoisomerase I.
J Biol Chem 1998 273 6050-6056
PubMed: 9497321
Lima CD
Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I.
Nature 1994 367 138-146
PubMed: 8114910
Bugreev DV
Structure and mechanism of action of type IA DNA topoisomerases.
Biochemistry (Mosc) 2009 74 1467-1481
PubMed: 20210704
Perry K
Biochemical characterization of an invariant histidine involved in Escherichia coli DNA topoisomerase I catalysis.
J Biol Chem 2002 277 13237-13245
PubMed: 11809772