18 research outputs found
Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum
Electrospray ionization mass spectrometry analysis of polyisoprenoid alcohols via Li+ cationization
Direct analysis of polyisoprenoids by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring offline time and sample-consuming derivatization techniques. We describe a simple ESI-MS approach for the direct analysis of polyisoprenoids using several dolichols and polyprenols with different chain sizes as proof-of-principle cases. Lithium iodide is used to promote cationization by intense formation of [M + Li](+) adducts. Thus, detection of polyisoprenoids with mass determination can be performed with high sensitivity (limit of detection [LOD] similar to 100 rho M), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenols permit investigation of their structure. Using ESI(Li+)-MS and ESI(Li+)-MS/MS analysis, we screened for polyprenol products of an octaprenyl pyrophosphate synthase of Plasmodium falciparum and dolichols in a complex mixture of compounds produced by Leishmania amazonensis and P. falciparum. (c) 2006 Elsevier Inc. All rights reserved.355218920
CHARGE association in a child with de novo chromosomal aberration 46,X,der(X)t(X;2)(p22.1;q33) detected by spectral karyotyping
Identification, molecular cloning and functional characterization of an octaprenyl pyrophosphate synthase in intra-erythrocytic stages of Plasmodium falciparum
Isoprenoids play important roles in all living organisms as components of structural cholesterol, steroid hormones in mammals, carotenoids in plants, and ubiquinones. Significant differences occur in the length of the isoprenic side chains of ubiquinone between different organisms, suggesting that different enzymes are involved in the synthesis of these side chains. Whereas in Plasmodiumfalciparum the isoprenic side chains of ubiquinone contain 7-9 isoprenic units, 10-unit side chains are found in humans. In a search for the P.falciparum enzyme responsible for the biosynthesis of isoprenic side chains attached to the benzoquinone ring of ubiquinones, we cloned and expressed a putative polyprenyl synthase. Polyclonal antibodies raised against the corresponding recombinant protein confirmed the presence of the native protein in trophozoite and schizont stages of P.falciparum. The recombinant protein, as well as P.falciparum extracts, showed an octaprenyl pyrophosphate synthase activity, with the formation of a polyisoprenoid with eight isoprenic units, as detected by reverse-phase HPLC and reverse-phase TLC, and confirmed by electrospray ionization and tandem MS analysis. The recombinant and native versions of the enzyme had similar Michaelis constants with the substrates isopentenyl pyrophosphate and farnesyl pyrophosphate. The recombinant enzyme could be competitively inhibited in the presence of the terpene nerolidol. This is the first report that directly demonstrates an octaprenyl pyrophosphate synthase activity in parasitic protozoa. Given the rather low similarity of the P. falciparum enzyme to its human counterpart, decaprenyl pyrophosphate synthase, we suggest that the identified enzyme and its recombinant version could be exploited in the screening of novel drugs.392111712
Non-random length distribution of individual telomeres in immunodeficiency, centromeric instability and facial anomalies syndrome, type I
The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum
Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate ( MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B-1) and pyridoxal (vitamin B-6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.27950517495175