The myo-inositol-1-phosphate synthase gene is essential in Trypanosoma brucei

2005 ◽  
Vol 33 (5) ◽  
pp. 983-985 ◽  
Author(s):  
K.L. Martin ◽  
T.K. Smith
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Essential Gene ◽  
Recombinant Expression ◽  
Conditional Knockout ◽  
Cellular Functions ◽  
Genome Database ◽  
Inositol 1 ◽  
Catalytically Active ◽  
Phosphate Synthase

The de novo synthesis of myo-inositol occurs via a two-step process: first, glucose 6-phosphate is converted into inositol 1-phosphate by an INO1 (myo-inositol-1-phosphate synthase; EC 5.5.1.4); then, it is dephosphorylated by an inositol monophosphatase. The myo-inositol can then be incorporated into PI (phosphatidylinositol), which is utilized in a variety of cellular functions, including the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. A putative INO1 was identified in the Trypanosoma brucei genome database and, by recombinant expression in Escherichia coli, was shown to be a catalytically active INO1. To investigate the importance of INO1, we created a conditional knockout, which, under non-permissive conditions, showed that INO1 is an essential gene in bloodstream form T. brucei and that the de novo synthesized myo-inositol is used for the formation of PI and GPI anchors.

scholarly journals Phosphatidylinositol synthesis is essential in bloodstream form Trypanosoma brucei

2006 ◽  
Vol 396 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Kirstee L. Martin ◽  
Terry K. Smith
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Essential Gene ◽  
Recombinant Expression ◽  
Protozoan Parasite ◽  
High Specificity ◽  
Bloodstream Form ◽  
Head Group ◽  
Double Knockout

PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasite's survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.

scholarly journals Myo-inositol content in pteridophytes and the isolation and characterization of L-myo-inositol-1-phosphate synthase from Diplopterygium glaucum

2006 ◽  
Vol 18 (2) ◽  
pp. 291-298 ◽  
Author(s):  
D. R. Chhetri ◽  
A. K. Mukherjee ◽  
J. Adhikari
Keyword(s):  
De Novo ◽  
Deae Cellulose ◽  
Normal Growth ◽  
Apparent Molecular Weight ◽  
Life Forms ◽  
Partial Purification ◽  
Ph Optimum ◽  
Inositol 1 ◽  
Isolation And Characterization ◽  
Phosphate Synthase

Myo-inositol is involved in normal growth and development of all living organisms and L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) is responsible for its de novo synthesis. This enzyme has been reported for a number of life forms including plants, animals and bacteria. In the present study free myo-inositol has been detected in the common pteridophytes found in the Darjeeling Himalayas and the enzyme, L-myo-inositol-1-phosphate synthase has been partially purified from Diplopterygium glaucum (Thunb.) Nakai. A crude homogenate from the reproductive pinnules of D. glaucum was subjected to streptomycin sulphate precipitation and 0-70% ammonium sulphate fractionation followed by successive chromatography through DEAE-cellulose, Hexylagarose and BioGel A-0.5m columns. This resulted in a partial purification of the enzyme of about 81-fold with 13.5% recovery. The pteridophytic MIPS specifically utilized D-glucose-6-phosphte and NAD+ as its substrate and co-factor, respectively. It shows a pH optimum between 7.0 and 7.5 while the temperature maximum was 30 °C. The enzyme activity was stimulated by NH4+, slightly inhibited by Na+, Ba2+ and Cd2+, and strongly inhibited by Li+, Zn2+ and Hg2+. EDTA, pCMB and some substrate isomers like glucose-1-phosphate, fructose-6-phosphte and galactose-6-phosphate were inhibitory to the enzyme. The apparent molecular weight of the native D. glaucum MIPS was determined to be approximately 171 kDa.

scholarly journals Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases

2008 ◽  
Vol 415 (1) ◽  
pp. 135-144 ◽  
Author(s):  
Federica Gibellini ◽  
William N. Hunter ◽  
Terry K. Smith
Keyword(s):  
Trypanosoma Brucei ◽  
Biochemical Characterization ◽  
De Novo ◽  
Carbon Chain ◽  
Surface Proteins ◽  
Membrane Phospholipids ◽  
Ethanolamine Kinase ◽  
Kennedy Pathway ◽  
Catalytically Active ◽  
Membrane Attachment

Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (glycerophosphoethanolamine) and GPCho (glycerophosphocholine). Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The Km values for ethanolamine and ATP were found to be 18.4±0.9 and 219±29 μM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a Km 80 times lower than that of ethanolamine. The Km values for choline, ethanolamine and ATP were 31.4±2.6 μM, 2.56±0.31 mM and 20.6±1.96 μM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.

scholarly journals Strategies for acquiring the phospholipid metabolite inositol in pathogenic bacteria, fungi and protozoa: making it and taking it

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1386-1396 ◽  
Author(s):  
Todd B. Reynolds
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Candida Albicans ◽  
Trypanosoma Brucei ◽  
Pathogenic Bacteria ◽  
De Novo ◽  
The Other ◽  
Inositol Monophosphatase ◽  
Essential Nutrient ◽  
Phosphate Synthase

myo-Inositol (inositol) is an essential nutrient that is used for building phosphatidylinositol and its derivatives in eukaryotes and even in some eubacteria such as the mycobacteria. As a consequence, fungal, protozoan and mycobacterial pathogens must be able to acquire inositol in order to proliferate and cause infection in their hosts. There are two primary mechanisms for acquiring inositol. One is to synthesize inositol from glucose 6-phosphate using two sequentially acting enzymes: inositol-3-phosphate synthase (Ino1p) converts glucose 6-phosphate to inositol 3-phosphate, and then inositol monophosphatase (IMPase) dephosphorylates inositol 3-phosphate to generate inositol. The other mechanism is to import inositol from the environment via inositol transporters. Inositol is readily abundant in the bloodstream of mammalian hosts, providing a source from which many pathogens could potentially import inositol. However, despite this abundance of inositol in the host, some pathogens such as the bacterium Mycobacterium tuberculosis and the protist parasite Trypanosoma brucei must be able to make inositol de novo in order to cause disease (M. tuberculosis) or even grow (T. brucei). Other pathogens such as the fungus Candida albicans are equally adept at causing disease by importing inositol or by making it de novo. The role of inositol acquisition in the biology and pathogenesis of the parasite Leishmania and the fungus Cryptococcus are being explored as well. The specific strategies used by these pathogens to acquire inositol while in the host are discussed in relation to each pathogen's unique metabolic requirements.

scholarly journals A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase fromPorteresia coarctata(Roxb.) Tateoka, a Halophytic Wild Rice

2004 ◽  
Vol 279 (27) ◽  
pp. 28539-28552 ◽  
Author(s):  
Manoj Majee ◽  
Susmita Maitra ◽  
Krishnarup Ghosh Dastidar ◽  
Sitakanta Pattnaik ◽  
Anirban Chatterjee ◽  
...  
Keyword(s):  
Wild Rice ◽  
Salt Tolerant ◽  
Inositol 1 ◽  
Phosphate Synthase
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