An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani.
DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes.
Das
Aditi
A
Molecular Parasitology Laboratory and Drug Design, Development and Molecular Modelling, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700032, India.
Mandal
Chhabinath
C
Dasgupta
Arindam
A
Sengupta
Tanushri
T
Majumder
Hemanta K
HK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012984:Software; D055672:Static Electricity; D013329:Structure-Activity Relationship
D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin