Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLK1) from Trypanosoma brucei

We have cloned and sequenced the gene for the glycerol kinase of Trypanosom a brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known gly cerol kinases of other organisms. It is 512 amino acids in length with a PT S1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal c ompartmentalization of this enzyme. Although Northern blot analysis reveale d higher mRNA levels in slender bloodstream forms than in the procyclic ins ect forms, specific glycerol kinase activities were found to be virtually i dentical in both life stages, Southern blot analysis suggested a single cop y gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli ena bled us to perform a kinetic analysis of this enzyme. In particular, we hav e been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded es sential for the survival of Trypanosoma brucei under anoxic conditions. Sin ce the unique spatial separation of glycolysis in the kinetoplastida impose s important consequences for the regulation of the energy metabolism in the se organisms, we discuss the observed differences between TbGLK1 and glycer ol kinases from other organisms in view of its physiological relevance.