Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: pyruvate, phosphate dikinase.
Trypanosomatids are parasitic protists that have an ATP-dependent glycolysis with no indication of PPi-dependent metabolism. Most of the glycolysis takes place in peroxisome-like organelles, the glycosomes. We characterized in Trypanosoma brucei a single-copy gene encoding a PPi-dependent enzyme, pyruvate, phosphate dikinase (PPDK), which was expressed functionally in Escherichia coli. Specific antibodies detected a 100-kDa protein in procyclic forms but not in mammalian forms of T. brucei, indicating a differential expression. Glycosomal localization of PPDK was determined by immunofluorescence analysis and was confirmed by Western blot analysis on glycosomal fractions by using anti-PPDK antibodies. Expression and localization of recombinant PPDKs in procyclic forms of T. brucei showed that the AKL motif at the C-terminal extremity of PPDK is necessary for glycosomal targeting. PPDK was detected in every trypanosomatid tested-Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Phytomonas, Crithidia and Leishmania-with a good correlation between amount of protein and enzymatic activity. The precise role of PPDK in trypanosomatid carbohydrate metabolism remains to be clarified.
Bringaud
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Laboratoire de Parasitologie Moléculaire, Université Victor Ségalène de Bordeaux II, France. bringaud@u-bordeaux2.fr
Baltz
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Baltz
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D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011772:Pyruvate, Orthophosphate Dikinase / Q000235:genetics* / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D015800:Protozoan Proteins; D011772:Pyruvate, Orthophosphate Dikinase