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 p athway sequestered inside peroxisome-like organelles: the glycosomes. So fa r, at least one enzyme of the pentose-phosphate pathway has been found to b e associated partially with glycosomes. Here, we describe how two genes fro m Trypanosoma brucei, coding for the first two enzymes of the pentose-phosp hate pathway, i.e. glucose-8 phosphate dehydrogenase and 6-phosphogluconola ctonase, were identified by in silico screening of trypanosome genome proje ct data bases. These genes were cloned and sequenced. Analysis of the lacto nase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream fo rm trypanosome (15% glycosomal and 85% cytosolic), However, the dehydrogena se sequence did not reveal any targeting signal, despite its localization i nside glycosomes, The corresponding enzymes have been overexpressed in Esch erichia coil and purified, and their biochemical characteristics have been determined.