Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei

- A putative NADP+-dependent type 1 IDH from Trpanosoma brucei brucei (TbIDH1) was identified.
- TbIDH1 contains a glycosomal targeting signal and shows high activity with both NADP+ and NAD+.
- Methods to express and purify large amounts of recombinant TbIDH1 for crystallographic study are discussed.
- Conditions and methods to crystallize recombinant TbIDH1 are presented.

Isocitrate dehydrogenases (IDHs) are metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate. Depending on the electron acceptor and subcellular localization, these enzymes are classified as NADP+-dependent IDH1 in the cytosol or peroxisomes, NADP+-dependent IDH2 and NAD+-dependent IDH3 in mitochondria. Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and Nagana disease in animals. Here, for the first time, a putative glycosomal T. brucei type 1 IDH (TbIDH1) was expressed in Escherichia coli and purified for crystallographic study. Surprisingly, the putative NADP+-dependent TbIDH1 has higher activity with NAD+ compared with NADP+ as electron acceptor, a unique characteristic among known eukaryotic IDHs which encouraged us to crystallize TbIDH1 for future biochemical and structural studies. Methods of expression and purification of large amounts of recombinant TbIDH1 with improved solubility to facilitate protein crystallization are presented.