Glycolytic pathway of the human malaria parasite Plasmodium falciparum: primary sequence analysis of the gene encoding 3-phosphoglycerate kinase and chromosomal mapping studies

Schuster

Proc. Natl. Acad. Sci. U.S.A.

1993

Ribonucleotide reductase (EC 1.17.4.1; RNR), a cell-cycle-regulated enzyme, catalyzes the ratelimiting step in the de novo synthesis ofdeoxyribonucleotides by the reduction of the corresponding ribonucleotides. The important role of the RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. However, nothing is known about this enzyme from the malaria parasite Plasmodium falciparum. We have isolated cDNA clones encoding both the large and small RNR subunits. the small subunit (R2) (3, 4). In E. coli and eukaryotic cells the Rl protomers, binding to substrates and allosteric effectors, have a M, of 83,000-87,000 (6). The R2 protomers, with a Mr of 37,000-45,000, contain ferric iron centers and a tyrosyl free radical essential for activity (7). The enzymesubstrate specificity and activity of RNR is regulated by a variety of nucleotides (2, 3). In this paper, we report the cloning and sequencing of P. falciparum Rl and the large and small R2 protomer cDNAs. ¶ The cell-cycle regulation of the subunit genes in synchronous intraerythrocytic cultures was also studied. The treatment of the parasite culture with an antisense oligonucleotide phosphorothioate directed against the R2 resulted in a significant growth inhibition; this suggests that the R2 is essential for cellular survival.MATERIALS AND METHODS Parasite Culture. P. falciparum strains HB3 and Dd2 were maintained in an erythrocyte culture as described by Trager and Jensen (7). If required, cultures were synchronized with two rounds of 5% D-sorbitol treatment (8).Nucleic Acid Isolation and Blot Analyses. The genomic DNA and RNA samples were isolated from saponin-lysed P. falciparum-infected erythrocytes by the SDS-proteinase K method (9) and the acidic guanidinium-phenol/chloroform method (10), respectively. Southern and RNA Blots were hybridized with random primer 32P-labeled RNR probes according to standard methods (9). A phosphorimager (Molecular

Section: Consensus --------Np ---------S ----Tnffe-rv--y----v ---------supporting

confidence: 73%

Section: Consensus --------Np ---------S ----Tnffe-rv--y----v ---------mentioning

confidence: 99%

See 1 more Smart Citation

Cloning and characterization of subunit genes of ribonucleotide reductase, a cell-cycle-regulated enzyme, from Plasmodium falciparum.

Schuster

Proc. Natl. Acad. Sci. U.S.A.

1993

“…Our genome analysis demonstrated the presence of a single pgk gene on chromosome 9 of this parasite, royalsocietypublishing.org/journal/rsob Open Biol. 10: 200302 in agreement with the result previously reported by [253]. The crystal structure of this glycolytic enzyme in the open conformation is similar to the structure of the monomeric PGKs from other organisms [47] (table 3).…”

Section: Apicomplexan Protistssupporting

confidence: 89%

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea

et al. 2020

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or ‘dead’ enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

“…Cloning and a high level of expression of genes encoding P. falciparum enzyme activities would permit studies on the structure and function of the parasiteÕs metabolic enzymes, eventually leading to identification of drugs that can selectively inhibit certain parasitic enzymes. The focus has been on cloning glycolytic pathway genes in P. falciparum because it is known that it relies on anaerobic glycolysis for energy production during the erythrocytic stages (Certa et al 1988; Kaslow and Hill 1990;Hicks et al 1991). However, its genome sequence revealed that many other biochemical pathways could also be reconstructed , suggesting the existence of yet uncharacterized pathways that may be essential for the parasiteÕs pathogenicity.…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of an alternative [lactate dehydrogenase-like] malate dehydrogenase in Plasmodium falciparum

Chan

Sim

2004

Parasitology Research

The catalysis of malate dehydrogenase (MDH) in Plasmodium falciparum (pfMDH) which involves NAD/NADH coupling is crucial for the parasite's pathogenicity. Primers were designed based on the P. falciparum genome resource, and these facilitated the cloning of a gene coding for pfMDH from a local clinical isolate. The DNA sequence of the cloned gene revealed an open-reading frame that encodes a protein of 313 amino acids. After induction in Escherichia coli BL21, enzyme assays of the expressed pfMDH purified by affinity chromatography exhibited significant enzyme activity of about 50 U/mg, where one unit (U) of enzyme activity is defined as the amount of enzyme oxidising 1 microol NADH/min. Based on its phylogenetic status amongst MDHs and lactate dehydrogenases (LDHs), the cloned gene was clearly defined as belonging to the NADH-dependent [LDH-like] MDHs. It is noteworthy that pfMDH harbours unique structural characteristics potentially useful for screening drugs specific for disabling parasitic enzymes.