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Identification of a Functional Type IA Topoisomerase, LdTopIIIβ, from Kinetoplastid Parasite Leishmania donovani.

Banerjee B, Sen N, Majumder HK - Enzyme Res (2011)

Bottom Line: Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid.A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite.Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Parasitology Laboratory, Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
DNA topoisomerases of kinetoplastids represent a family of DNA processing enzymes that essentially solve the topological problems not only in nuclear DNA but also in kinetoplast DNA. We have, for the first time, identified a Leishmania donovani homologue of bacterial and eukaryotic IA type of topoisomerase III protein and termed as LdTopIIIβ. Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid. A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite. Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

No MeSH data available.


Amino acid sequence alignment. Sequence of LdTopIIIβ (Ld) was aligned with the amino acid sequences of H. sapiens topoisomerase IIIβ (Hs), topoisomerase III from S. cerevisiae (Sc) and S. pombe (Sp) using CLUSTAL W. The amino acids are numbered on the top of the sequences. Active site motifs and other important conserved and identical residues are depicted in red. Green and blue indicate strongly similar and weakly similar amino acids, respectively.
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fig1: Amino acid sequence alignment. Sequence of LdTopIIIβ (Ld) was aligned with the amino acid sequences of H. sapiens topoisomerase IIIβ (Hs), topoisomerase III from S. cerevisiae (Sc) and S. pombe (Sp) using CLUSTAL W. The amino acids are numbered on the top of the sequences. Active site motifs and other important conserved and identical residues are depicted in red. Green and blue indicate strongly similar and weakly similar amino acids, respectively.

Mentions: One of the two topoisomerase III genes present in L. major geneDB is 2601 bp (LmjF28.1780) and encodes a 95 kDa predicted protein. The other topoisomerase III ORF (LmjF36.3200) is 2844 bp, and encodes a 104 kDa predicted protein. Topoisomerase III gene with 2601 bp was PCR amplified from the genomic DNA of L. donovani, cloned and sequenced (GeneBank accession number GQ499197). Blast analysis of the sequence confirmed the topoisomerase III lineage of the protein and henceforth referred as LdTopIIIβ. The alignment of LdTopIIIβ with S. cerevisiae and S. pombe topoisomerase III and human topoisomerase III is shown in Figure 1. The active site tyrosine is located at the 327 position within a highly conserved GYISYPRTES sequence. The protein has 46.22% identity and 76.09% similarity with human topoisomerase IIIβ. It contains seven CXXC sequences instead of eight found in other topoisomerase IIIβ proteins. The intervening spacers are also highly conserved. Glycine (G) and arginine (R) rich clusters at the C-terminus end, which is another hallmark of topoisomerase IIIβ, are also present. It has a continuous stretch of 19 G and R residues in the C-terminus. Three-dimensional structure generated by Swiss Prot has been shown in Figure 2(a). Figure 2(b) shows the magnified view of the active site. The conserved amino acid residues are represented in ball and stick format and have been labeled. Homology comparisons of LdTopIIIβ with other IA type of topoisomerases have been provided in Table 1, which strongly indicates its topoisomerase III lineage.


Identification of a Functional Type IA Topoisomerase, LdTopIIIβ, from Kinetoplastid Parasite Leishmania donovani.

Banerjee B, Sen N, Majumder HK - Enzyme Res (2011)

Amino acid sequence alignment. Sequence of LdTopIIIβ (Ld) was aligned with the amino acid sequences of H. sapiens topoisomerase IIIβ (Hs), topoisomerase III from S. cerevisiae (Sc) and S. pombe (Sp) using CLUSTAL W. The amino acids are numbered on the top of the sequences. Active site motifs and other important conserved and identical residues are depicted in red. Green and blue indicate strongly similar and weakly similar amino acids, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102327&req=5

fig1: Amino acid sequence alignment. Sequence of LdTopIIIβ (Ld) was aligned with the amino acid sequences of H. sapiens topoisomerase IIIβ (Hs), topoisomerase III from S. cerevisiae (Sc) and S. pombe (Sp) using CLUSTAL W. The amino acids are numbered on the top of the sequences. Active site motifs and other important conserved and identical residues are depicted in red. Green and blue indicate strongly similar and weakly similar amino acids, respectively.
Mentions: One of the two topoisomerase III genes present in L. major geneDB is 2601 bp (LmjF28.1780) and encodes a 95 kDa predicted protein. The other topoisomerase III ORF (LmjF36.3200) is 2844 bp, and encodes a 104 kDa predicted protein. Topoisomerase III gene with 2601 bp was PCR amplified from the genomic DNA of L. donovani, cloned and sequenced (GeneBank accession number GQ499197). Blast analysis of the sequence confirmed the topoisomerase III lineage of the protein and henceforth referred as LdTopIIIβ. The alignment of LdTopIIIβ with S. cerevisiae and S. pombe topoisomerase III and human topoisomerase III is shown in Figure 1. The active site tyrosine is located at the 327 position within a highly conserved GYISYPRTES sequence. The protein has 46.22% identity and 76.09% similarity with human topoisomerase IIIβ. It contains seven CXXC sequences instead of eight found in other topoisomerase IIIβ proteins. The intervening spacers are also highly conserved. Glycine (G) and arginine (R) rich clusters at the C-terminus end, which is another hallmark of topoisomerase IIIβ, are also present. It has a continuous stretch of 19 G and R residues in the C-terminus. Three-dimensional structure generated by Swiss Prot has been shown in Figure 2(a). Figure 2(b) shows the magnified view of the active site. The conserved amino acid residues are represented in ball and stick format and have been labeled. Homology comparisons of LdTopIIIβ with other IA type of topoisomerases have been provided in Table 1, which strongly indicates its topoisomerase III lineage.

Bottom Line: Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid.A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite.Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Parasitology Laboratory, Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
DNA topoisomerases of kinetoplastids represent a family of DNA processing enzymes that essentially solve the topological problems not only in nuclear DNA but also in kinetoplast DNA. We have, for the first time, identified a Leishmania donovani homologue of bacterial and eukaryotic IA type of topoisomerase III protein and termed as LdTopIIIβ. Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid. A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite. Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

No MeSH data available.