Catalytic Site Atlas
LITERATURE entry for 1a41
|E.C. name||DNA topoisomerase|
|Species||Vaccinia virus (Virus)|
E.C. Number (IntEnz)
|CSA Homologues of 1a41||
CSA Entries With UniProtID
CSA Entries With EC Number
|MACiE Entry ||M0232|
|Introduction||DNA topoisomerase IB (TopIB), isolated from the vaccinia virus, catalyses the relaxation of negative and positive supercoiling in DNA. Unlike other type I topoisomerases TopIB exhibits specificity for cleavage at a consensus sequence: 5'-(T/C)CCTT-3' in the scissile strand with cleavage occuring after the last base. The binding of DNA causes conformational changes in the enzyme and catalytic residues are recruited to form the active site. The reaction is independent of metal cations and ATP hydrolysis.
|Mechansim||Tyr274 is the nucleophile for in-line displacement at the DNA phosphate group with the 5'-hydroxyl as the leaving group. The transition state is stabilised by Arg130 binding to one non-bridging oxygen, Arg223 and His265 binding to the other non-bridging oxygen, and Arg223 and Lys167 binding to the 5'-hydroxyl leaving group. Either Arg223 or a water molecule is involved in general base catalysis with respect to Tyr274 while Lys167 and Arg130 are involved in general acid catalysis with respect to the 5'-hydroxyl leaving group. Rotation of the DNA-enzyme intermediate around the uncleaved strand then occurs, relaxing the supercoiled DNA. The 5'-hydroxyl is then the nucleophile for in-line displacement at the DNA-enzyme intermediate. The transition state is stabilised in a similar way to that of DNA cleavage. Tyr274 is the leaving group and is protonated by Arg223/water.
Catalytic Sites for 1a41
| Annotated By Reference To The Literature - Site 2 (Perform Site Search)|
|Residue||Chain||Number||UniProtKB Number||Functional Part||Function||Target||Description|
|Tyr||A||274||274||macie:sideChain||Tyr274 is the nucleophile for attack on the phosphate group during DNA cleavage. The pKa of the phenol is thought to be lowered by Arg223 to make it a better nucleophile. Arg223 may also act as a base to deprotonate Tyr274, or a water molecule could fill this role. Tyr274 is the leaving group during DNA ligation and is protonated by Arg223/water.|
|Arg||A||223||223||macie:sideChain||Arg223 stabilises the transition state of DNA cleavage by coordinating one of the phosphate non-bridging oxygens. It is though to lower the pKa of Tyr274 and may be involved in the deprotonation of Tyr274 during cleavage. If the latter is the case then it would presumably act as an acid during DNA ligation, as well as stabilising the transition state.|
|His||A||265||265||macie:sideChain||His265 stabilises the transition states of DNA cleavage and ligation by coordinating to one of the phosphate non-bridging oxygens.|
|Lys||A||167||167||macie:sideChain||Lys167 coordinates the 5'-hydroxyl leaving group during DNA cleavage and is thought to be involved in general acid catalysis. Although the exact mechanism of protonation is uncertain, there is evidence to suggest that Arg130 and Lys167 act in a concerted fashion in the protonation of the O-5'.|
Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.
Nucleic Acids Res 1997 25 3001-3008
The structure of the transition state of the heterodimeric topoisomerase I of Leishmania donovani as a vanadate complex with nicked DNA.
J Mol Biol 2006 357 1202-1210
Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis.
J Biol Chem 1997 272 8263-8269
Stereochemical outcome and kinetic effects of Rp- and Sp-phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase.
Biochemistry 2000 39 5561-5572
Structural basis for specificity in the poxvirus topoisomerase.
Mol Cell 2006 23 343-354
Chemical and traditional mutagenesis of vaccinia DNA topoisomerase provides insights to cleavage site recognition and transesterification chemistry.
J Biol Chem 2008 283 16093-16103
Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.
Proc Natl Acad Sci U S A 1989 86 9793-9797
Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases.
Cell 1998 92 841-850