Cloning of a pyruvate phosphate dikinase from Trypanosoma cruzi.

ABSTRACT

We have cloned and characterised a gene that encodes a putative pyruvate phosphate dikinase (PPDK) from Trypanosoma cruzi, an enzyme that catalyses the reversible conversion of phosphoenolpyruvate to pyruvate. PPDK is absent in mammalian cells, but has been found in a wide variety of other organisms, including plants and bacteria. In T. cruzi, two genes (PPDK1 and PPDK2) are present in a tandem array localised on a 1 Mbp chromosome. Northern and Western blot analyses indicates that PPDK is expressed as a 100-kDa protein in epimastigote, amastigote and trypomastigote forms. PPDK1 and PPDK2 encode an identical protein of 100.8 kDa with a C-terminal extension ending with the sequence AKL, a signal for glycosomal import. Both T. cruzi and T. brucei enzymes possess a 23-residue insertion, that is absent in other PPDKs. A three-dimensional alignment with the crystal structure of the enzyme from Clostridium symbiosum predicts that this insertion is located on the surface of the nucleotide-binding domain. Phylogenetic studies indicate that bacterial and protist PPDKs cluster as a separate group from those of plants. The evolutionary implications and possible role of this enzyme in T. cruzi is discussed.