Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase.
Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.
Duffieux
F
F
Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and the Laboratory of Biochemistry, Université Catholique de Louvain, Avenue Hippocrate 74, 1200 Brussels, Belgium.
Van Roy
J
J
Michels
P A
PA
Opperdoes
F R
FR
D000595:Amino Acid Sequence; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017125:Genes, Protozoan; D018503:Genome, Protozoan; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010427:Pentose Phosphate Pathway; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase; D002265:Carboxylic Ester Hydrolases; C022363:6-phosphogluconolactonase