Erratum to “Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method”[Mol. Biochem. Parasitol. 137 (2004) 13–21]

Tonkin, Christopher J.; Vandooren, G; Spurck, Timothy P.; Struck, Nicole S.; Good, Robert T.; Handman, Emanuela; Cowman, Alan; McFadden, G. I.

doi:10.1016/s0166-6851(04)00249-x

Cited by 4 publications

(3 citation statements)

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“…Localization of PfPBGD-To study the localization of PfPBGD in P. falciparum the method described by Tonkin et al (25) was followed. Polyclonal antibodies were raised against ⌬PfPBGD in rabbit and IgG was purified using protein A Sepharose chromatography.…”

Section: Methodsmentioning

confidence: 99%

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Nagaraj

Arumugam

Bulusu

et al. 2008

Journal of Biological Chemistry

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The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (⌬PfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (URO-GEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, ⌬PfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a ⌬PfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than ⌬PfPBGD. More interestingly, ⌬PfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to URO-GEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to URO-GEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.Detailed studies of the metabolic pathways in the malarial parasite hold promise for the identification of new antimalarial drug targets (1, 2). Earlier studies in this laboratory had shown that the malarial parasite synthesizes heme de novo, despite acquiring heme from the host red cell hemoglobin in the intraerythrocytic stage. It has been shown that Plasmodium falciparum contains ␦-aminolevulinate dehydratase (ALAD) 2 of dual origin: one species encoded by the parasite genome (PfALAD) and another imported from the host red cell. Inhibition of ALAD activity in the parasite, the second enzyme of the pathway with a specific inhibitor, succinylacetone, leads to inhibition of heme synthesis and death of the parasite, indicating the potential of the pathway as a drug target (3, 4).Heme biosynthesis from glycine and succinyl-CoA involves eight different steps and the genes for all the enzymes of the pathway, with the exception of uroporphyrinogen III synthase (UROS, hemD) have been located on the parasite genome (5). However, a hemD orthologue has been identified in Toxoplasma gondii and is expected to be targeted to the apicoplast, a chloroplast-like organelle in the parasite (6). Based mostly on bioinformatics-based predictions, some e...

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Section: Methodsmentioning

confidence: 99%

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Nagaraj

Arumugam

Bulusu

et al. 2008

Journal of Biological Chemistry

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“…Immunofluorescence assays using confocal microscopy Immunofluorescence assay (IFA) was performed as described earlier [34]. Cells were washed in PBS and fixed in solution using 4 % w/v paraformaldehyde and 0.0075 % w/v glutaraldehyde in PBS for 25 min.…”

Section: Production Of Polyclonal Antibodies and Expression Analysismentioning

confidence: 99%

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase

Jain

Kikuchi

Oshima

et al. 2014

J Struct Funct Genomics

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Aminoacyl-tRNA synthetases (aaRSs) drive protein translation in cells and hence these are essential enzymes across life. Inhibition of these enzymes can halt growth of an organism by stalling protein translation. Therefore, small molecule targeting of aaRS active sites is an attractive avenue from the perspective of developing anti-infectives. Febrifugine and its derivatives like halofuginone (HF) are known to inhibit prolyl-tRNA synthetase of malaria parasite Plasmodium falciparum. Here, we present functional and crystallographic data on P. falciparum prolyl-tRNA synthetase (PfPRS). Using immunofluorescence data, we show that PfPRS is exclusively resident in the parasite cytoplasm within asexual blood stage parasites. The inhibitor HF interacts strongly with PfPRS in a non-competitive binding mode in presence or absence of ATP analog. Intriguingly, the two monomers that constitute dimeric PfPRS display significantly different conformations in their active site regions. The structural analyses presented here provide a framework for development of febrifugine derivatives that can seed development of new anti-malarials.

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“…An immunofluorescence assay (IFA) was performed as described previously (Tonkin et al, 2004). Cells were washed in PBS and fixed in solution using 4%(w/v) paraformaldehyde and 0.0075%(w/v) glutaraldehyde in PBS for 25 min.…”

Section: Immunofluorescence Assays Using Confocal Microscopymentioning

confidence: 99%

Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development

Khan

Garg

Camacho

et al. 2013

Acta Crystallogr D Biol Crystallogr

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Aminoacyl-tRNA synthetases are essential enzymes that transmit information from the genetic code to proteins in cells and are targets for antipathogen drug development. Elucidation of the crystal structure of cytoplasmic lysyl-tRNA synthetase from the malaria parasite Plasmodium falciparum (PfLysRS) has allowed direct comparison with human LysRS. The authors' data suggest that PfLysRS is dimeric in solution, whereas the human counterpart can also adopt tetrameric forms. It is shown for the first time that PfLysRS is capable of synthesizing the signalling molecule Ap4a (diadenosine tetraphosphate) using ATP as a substrate. The PfLysRS crystal structure is in the apo form, such that binding to ATP will require rotameric changes in four conserved residues. Differences in the active-site regions of parasite and human LysRSs suggest the possibility of exploiting PfLysRS for selective inhibition. These investigations on PfLysRS further validate malarial LysRSs as attractive antimalarial targets and provide new structural space for the development of inhibitors that target pathogen LysRSs selectively.

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Erratum to “Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method”[Mol. Biochem. Parasitol. 137 (2004) 13–21]

Cited by 4 publications

References 0 publications

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase

Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development

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