1. Transketolase in Trypanosoma brucei
- Author
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Vincent P. Alibu, Michael P. Barrett, Jean-Charles Portais, Frédéric Bringaud, Jane Hubert, Sabine A. Stoffel, Charles Ebikeme, M. Ernst Schweingruber, Pev Biotech AG, Partenaires INRAE, University of Glasgow, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université de Bordeaux Ségalen [Bordeaux 2], Universität Bern- University of Bern [Bern], Swiss National Foundation, Wellcome Trust, Agence Nationale de la Recherche (ANR) [ANR-MIME2007], BBSRC-ANR, and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
[SDV]Life Sciences [q-bio] ,Trypanosoma brucei brucei ,Mutant ,Gene Expression ,Protein Sorting Signals ,Transketolase ,Biology ,Trypanosoma brucei ,METABOLISM ,Microbodies ,03 medical and health sciences ,chemistry.chemical_compound ,Cytosol ,[SDV.IDA]Life Sciences [q-bio]/Food engineering ,Escherichia coli ,Metabolic profiling ,BLOOD-STREAM FORMS ,[SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering ,OXIDATIVE STRESS ,PENTOSE-PHOSPHATE PATHWAY ,LEISHMANIA-MEXICANA ,Molecular Biology ,Gene ,Peroxisomal targeting signal ,Pentose phosphate pathway ,030304 developmental biology ,Pentosephosphates ,0303 health sciences ,THIAMIN CATALYSIS ,Gene Expression Profiling ,030302 biochemistry & molecular biology ,Wild type ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,LIFE-CYCLE STAGES ,Kinetics ,Protein Transport ,Sedoheptulose ,chemistry ,Biochemistry ,ESCHERICHIA-COLI ,Ttypanosoma brucei ,Transketolase activity ,Parasitology ,Ribosemonophosphates ,DIPHOSPHATE-DEPENDENT ENZYME ,MESSENGER-RNA - Abstract
International audience; A single copy gene, encoding a protein highly similar to transketolase from other systems, was identified in the Trypanosoma brucei genome. The gene was expressed in E. coil and the purified protein demonstrated transketolase activity with Km values of 0.2 mM and 0.8 mM respectively for xylulose 5-phosphate and ribose 5-phosphate. A peroxisomal targeting signal (PTS-1) present at the C-terminus of the protein suggested a glycosomal localisation. However, subcellular localisation experiments revealed that while the protein was present in glycosomes it was found mainly within the cytosol and thus has a dual localisation. Transketolase activity was absent from the long slender bloodstream form of the parasite and the protein was not detectable in this life cycle stage, with the RNA present only at low abundance, indicating a strong differential regulation, being present predominantly in the procyclic form. The gene was knocked out from procyclic T. brucei and transketolase activity was lost but no growth phenotype was evident in the null mutants. Metabolite profiling to compare wild type and TKT null mutants revealed substantial increases in transketolase substrate metabolites coupled to loss of sedoheptulose 7-phosphate, a principal product of the transketolase reaction. (C) 2011 Elsevier B.V. All rights reserved.
- Published
- 2011