CLONING AND CHARACTERIZATION OF THE NAD-LINKED GLYCEROL-3-PHOSPHATE DEHYDROGENASES OF TRYPANOSOMA-BRUCEI-BRUCEI AND LEISHMANIA-MEXICANA MEXICANA AND EXPRESSION OF THE TRYPANOSOME ENZYME IN ESCHERICHIA-COLI

A polyclonal antiserum raised against the purified glycosomal glycerol -3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression l ibrary. This cDNA was subsequently used to obtain genomic clones conta ining glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the ge nes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37 651 Da and a calculated net charge of +8. Using t he T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate d ehydrogenase gene was also identified in a genomic library of Leishman ia mexicana mexicana. The L.m. mexicana gene codes for a polypeptide o f 365 amino acids, with a molecular mass of 39 140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 6 3% identical and carry a type-1 peroxisomal targeting signal (PTSI) SK M and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis sho wed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activ ity co-fractionated both with mitochondria and with glycosomes. This i s not the case in T. brucei, where the enzyme is predominantly glycoso mal. The two trypanosomatid sequences resemble their prokaryotic homol ogues (32-36%) more than their eukaryotic counterparts (25-31%) and ca rry typical prokaryotic signatures. The possible reason for this proka ryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase i s discussed.