Crystal Structures of SCP2 Thiolases of Trypanosomatidae, Human Pathogens Causing Widespread Tropical Diseases The Importance for Catalysis of the Cysteine of the Unique Hdcf Loop.

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.</jats:p

An Interface Point-mutation Variant of Triosephosphate Isomerase Is Compactly Folded and Monomeric At Low-protein Concentrations

Wild-type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer, The interface residue His-47 of wtTIM has been mutated into an asparagine, Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml, H47N has been characterised at a protein concentration,where it is predominantly a monomer, Circular dichroism measurements in the near-UV and far-UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give T-m-values of 41 degrees C for wtTIM, whereas the T-m-value for the monomeric form of H47N is approximately 7 degrees C lower