title: Transketolase in Trypanosoma brucei.
abstract: 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. coli and the purified protein demonstrated transketolase activity with K(m) values of 0.2mM and 0.8mM 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.
authors.affiliation: Pevion Biotech AG, Worblentalstrasse 32, CH-3063 Ittigen/BE, Switzerland.
authors.forename: Sabine A
authors.initials: SA
authors.lastname: Stoffel
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Alibu
authors.forename: Jane
authors.initials: J
authors.lastname: Hubert
authors.forename: Charles
authors.initials: C
authors.lastname: Ebikeme
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bringaud
authors.forename: M Ernst
authors.initials: ME
authors.lastname: Schweingruber
authors.forename: Michael P
authors.initials: MP
authors.lastname: Barrett
mesh_terms: D003600:Cytosol / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D007700:Kinetics; D008830:Microbodies / Q000737:chemistry; D010428:Pentosephosphates / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism; D014174:Transketolase / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D010428:Pentosephosphates; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; C031625:xylulose-5-phosphate; D014174:Transketolase