A novel active DNA topoisomerase I in Leishmania donovani

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.     

N-terminal region of the large subunit of Leishmania donovani bisubunit topoisomerase I is involved in DNA relaxation and interaction with the smaller subunit

Leishmania donovani topoisomerase I is an unusual bisubunit enzyme. We have demonstrated earlier that the large and small subunit could be reconstituted in vitro to show topoisomerase I activity. We extend our biochemical study to evaluate the role of the large subunit in topoisomerase activity. The large subunit (LdTOP1L) shows a substantial degree of homology with the core DNA binding domain of the topoisomerase IB family. Two N-terminal truncation constructs, LdTOP1Delta39L (lacking amino acids 1-39) and LdTOP1Delta99L (lacking amino acids 1-99) of the large subunit were generated and mixed with intact small subunit (LdTOP1S). Our observations reveal that residues within amino acids 1-39 of the large subunit have significant roles in modulating topoisomerase I activity (i.e. in vitro DNA relaxation, camptothecin sensitivity, cleavage activity, and DNA binding affinity). Interestingly, the mutant LdTOP1Delta99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Delta99L was unable to pull down glutathione S-transferase-LdTOP1S in an Ni(2+)-nitrilotriacetic acid co-immobilization experiment. For further analysis, we co-expressed LdTOP1L and LdTOP1S in Escherichia coli BL21(DE3)pLysS cells. The lysate shows topoisomerase I activity. Immunoprecipitation revealed that LdTOP1L could interact with LdTOP1S, indicating the subunit interaction in bacterial cells, whereas immunoprecipitation of bacterial lysate co-expressing LdTOP1Delta99L and LdTOP1S reveals that LdTOP1Delta99L was significantly deficient at interacting with LdTOP1S to reconstitute topoisomerase I activity. This study demonstrates that heterodimerization between the large and small subunits of the bisubunit enzyme appears to be an absolute requirement for topoisomerase activity. The residue within amino acids 1-39 from the N-terminal end of the large subunit regulates DNA topology during relaxation by controlling noncovalent DNA binding or by coordinating DNA contacts by other parts of the enzyme.     

Characterization of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase.

In order to clone the gene encoding a type I DNA topoisomerase from Leishmania donovani, a PCR-amplified DNA fragment obtained with degenerate oligodeoxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 amino acid residues was isolated. A substantial part of the protein shares a significant degree of homology with the sequence of other known members of the IB topoisomerase family, in a highly conserved region of these enzymes termed the core domain. However, homology is completely lost after this conserved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coli did not show any relaxation activity in vitro and was unable to complement a mutant deficient in topoisomerase I activity. The results of Southern blot experiments strongly suggested that the cloned gene was not a pseudogene. Northern analysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is preferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.     

An insight into the mechanism of inhibition of unusual bi-subunit topoisomerase I from Leishmania donovani by 3,3'-di-indolylmethane, a novel DNA topoisomerase I poison with a strong binding affinity to the enzyme

DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.     

Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani

Leishmania donovani topoisomerase I is an unusual bi-subunit enzyme. The activity of the enzyme has been detected when the genes of the individual subunits were co-expressed in yeast [J. Biol. Chem. 278 (2003) 3521]. Here, we report for the first time, the in vitro reconstitution of the two recombinant proteins, LdTOP1L and LdTOP1S, corresponding to the large and small subunits and localization of the active enzyme in both the nucleus and kinetoplast. The proteins were purified from bacterial extract and the activity was measured by plasmid DNA relaxation assay. LdTOP1L and LdTOP1S form a direct 1:1 heterodimer complex through protein-protein interaction. Under standard relaxation assay condition (50 mM KCl and 10 mM Mg(2+)), reconstituted enzyme (LdTOP1LS) showed reduced processivity as well as 2-fold reduced affinity for DNA compared to eukaryotic monomeric rat liver topoisomerase I (RLTOP1). Cleavage assay at various salt concentrations reveals that Camptothecin (CPT) enhanced the formation of "cleavable complex" at low salt. Interaction between the two subunits leading to the formation of an active complex could be explored as an insight for development of new therapeutic agents with specific selectivity.     

Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovani

The amino acid sequences of the C-terminal domain (CTD) of the type II DNA topoisomerases are divergent and species specific as compared with the highly conserved N-terminal and central domains. A set of C-terminal deletion mutants of Leishmania donovani topoisomerase II was constructed. Removal of more than 178 amino acids out of 1236 amino acid residues from the C-terminus inactivates the enzyme, whereas removal of 118 amino acids or less has no apparent effect on the ability of the parasite enzyme to complement a temperature-sensitive mutation of the Saccharomyces cerevisiae topoisomerase II gene. Deletion analysis revealed a potent nuclear localization signal (NLS) within the amino acid residues 998–1058. Immunomicroscopy results suggest that the removal of an NLS in the CTD is likely to contribute to the physiological dysfunction of these proteins. Modeling of the LdTOP2 based on the crystal structure of the yeast type II DNA topoisomerase showed that the parasite protein assumes a structure similar to its yeast counterpart harboring all the conserved residues in a structurally similar position. However, a marked difference in electrostatic potential was found in a span of 60 amino acid residues (998–1058), which also do not have any homology with topoisomerase II sequences. Such significant differences can be exploited by the structure-based design of selective inhibitors using the structure of the Leishmania enzyme as a template.     

ATP independent type IB topoisomerase of Leishmania donovani is stimulated by ATP: an insight into the functional mechanism

Most type IB topoisomerases do not require ATP and Mg(2+) for activity. However, as shown previously for vaccinia topoisomerase I, we demonstrate that ATP stimulates the relaxation activity of the unusual heterodimeric type IB topoisomerase from Leishmania donovani (LdTOP1L/S) in the absence of Mg(2+). The stimulation is independent of ATP hydrolysis but requires salt as a co-activator. ATP binds to LdTOP1L/S and increases its rate of strand rotation. Docking studies indicate that the amino acid residues His93, Tyr95, Arg188 and Arg190 of the large subunit may be involved in ATP binding. Site directed mutagenesis of these four residues individually to alanine and subsequent relaxation assays reveal that the R190A mutant topoisomerase is unable to exhibit ATP-mediated stimulation in the absence of Mg(2+). However, the ATP-independent relaxation activities of all the four mutant enzymes remain unaffected. Additionally, we provide evidence that ATP binds LdTOP1L/S and modulates the activity of the otherwise ATP-independent enzyme. This study establishes ATP as an activator of LdTOP1L/S in the absence of Mg(2+).     

A unique modification of the eukaryotic initiation factor 5A shows the presence of the complete hypusine pathway in Leishmania donovani

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N(€)-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of α-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A ~42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed ~40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite.     

Structure and expression of a tandem gene pair in Leishmania donovani that encodes a protein structurally homologous to eucaryotic cation-transporting ATPases.

An oligonucleotide probe was used to clone a cation-transporting ATPase gene from the genome of Leishmania donovani. The nucleotide sequence of the gene contained a 2,922-base-pair open reading frame that was predicted to encode a 107,406-dalton protein composed of 974 amino acids. The predicted L. donovani protein contained all the structural and functional domains expected to be present in a cation-transporting ATPase of the aspartyl phosphate class. The nucleotide sequence encoding the ATPase gene was duplicated in tandem in the parasite genome. Partial sequenation of the second member of the tandem repeat, which lay 2 kilobase pairs downstream of the ATPase gene, indicated that it was either identical to the first gene or very closely related to it. RNA homologous to either the ATPase gene or its adjacent relative was 5 kilobases in size and was approximately equally abundant in both promastigote and amastigote forms of the organism.     

A glutathione-specific aldose reductase of Leishmania donovani and its potential implications for methylglyoxal detoxification pathway

Methylglyoxal is mainly catabolized by two major enzymatic pathways. The first is the ubiquitous detoxification pathway, the glyoxalase pathway. In addition to the glyoxalase pathway, aldose reductase pathway also plays a crucial role in lowering the levels of methylglyoxal. The gene encoding aldose reductase (ALR) has been cloned from Leishmania donovani, a protozoan parasite causing visceral leishmaniasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 855 bp encoding a putative protein of 284 amino acids with a calculated molecular mass of 31.7 kDa and a predicted isoelectric point of 5.85. The sequence identity between L. donovani ALR (LdALR) and mammals and plants is only 36-44%. The ORF is a single copy gene. A protein with a molecular mass that matched the estimated approximately 74 kDa according to the amino acid composition of LdALR with a maltose binding tag present at its N-terminal end was induced by heterologous expression of LdALR in Escherichia coli. In the presence of glutathione, recombinant LdALR reduced methylglyoxal with a K(m) of approximately 112 microM. Comparative structural analysis of the human ALR structure with LdALR model suggests that the active site anchoring the N-terminal end of the glutathione is highly conserved. However, the C-terminal end of the glutathione backbone is expected to be exposed in LdALR, as the residues anchoring the C-terminal end of the glutathione backbone come from the three loop regions in human, which are apparently shortened in the LdALR structure. Thus, the computational analysis provides clues about the expected mode of glutathione binding and its interactions with the protein. This is the first report of the role of an ALR in the metabolic disposal of methylglyoxal in L. donovani and of thiol binding to a kinetoplastid aldose reductase.     

'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.     

Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24.

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.     

Characterization of cDNA encoding the mouse DNA topoisomerase II that can complement the budding yeast top2 mutation.

Several cDNA clones encoding mouse DNA topoisomerase II were obtained from a mouse spermatocyte cDNA library and the entire coding sequence of the gene was determined. The mouse DNA topoisomerase II consists of 1528 amino acids with a molecular weight of 173 kDa. It shares significant homologies with the other eucaryotic enzymes, although species-specific sequences are observed in their highly charged C-terminal regions. The complete mouse TOP2 cDNA was put under yeast GAL1 promoter and examined for complementation of top2ts mutation in S.cerevisiae. We found that the cloned mouse gene could rescue the temperature-sensitive top2ts mutation, depending on its induction by galactose. The functional expression of the mouse DNA topoisomerase II in yeast was further confirmed by enzymatic assays and by immunological methods with antibodies specific for the mouse enzyme.     

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5 and 3 splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg²⁺- and ATP-dependent manner, and this activity was inhibited by 4-(9-acridinylamino)-3-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII ^ts yeast mutant.     

African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases.

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.