Glycerol kinase of Trypanosoma brucei - Cloning, molecular characterization and mutagenesis

Trypanosoma brucei contains two tandemly arranged genes for glycerol kinase . The downstream gene was analysed in detail. It contains an ORF for a poly peptide of 512 amino acids. The polypeptide has a calculated molecular mass of 56 363 Da and a pI of 8.6. Comparison of the T. brucei glycerol kinase amino-acid sequence with the glycerol kinase sequences available in databas es revealed positional identities of 39.0-50.4%. The T. brucei glycerol kin ase gene was overexpressed in Escherichia coli cells and the recombinant pr otein obtained was purified and characterized biochemically. Its kinetic pr operties with regard to both the forward and reverse reaction were measured . The values corresponded to those determined previously for the natural gl ycerol kinase purified from the parasite, and confirmed that the apparent K -m values of the trypanosome enzyme for its substrates are relatively high compared with those of other glycerol kinases. Alignment of the amino-acid sequences of T. brucei glycerol kinase and other eukaryotic and prokaryotic glycerol kinases, as well as inspection of the available three-dimensional structure of E. coli glycerol kinase showed that most residues of the magn esium-, glycerol- and ADP-binding sites are well conserved in T. brucei gly cerol kinase. However, a number of remarkable substitutions was identified, which could be responsible for the low affinity for the substrates. Most s triking is amino-acid Ala137 in T. brucei glycerol kinase; in all other org anisms a serine is present at the corresponding position. We mutated Ala137 of T. brucei glycerol kinase into a serine and this mutant glycerol kinase was over-expressed and purified. The affinity of the mutant enzyme for its substrates glycerol and glycerol 3-phosphate appeared to be 3.1-fold to 3. 6-fold higher than in the wild-type enzyme. Part of the glycerol kinase gen e comprising this residue 137 was amplified in eight different kinetoplasti d species and sequenced. Interestingly, an alanine occurs not only in T. br ucei, but also in other trypanosomatids which can convert glucose into equi molar amounts of glycerol and pyruvate: T. gambiense, T. equiperdum and T. evansi. In trypanosomatids with no or only a limited capacity to produce gl ycerol, a hydroxy group-containing residue is found as in all other organis ms: T. vivax and T. congolense possess a serine while Phytomonas sp., Leish mania brasiliensis and L. mexicana have a threonine.