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1.
Gene ; 257(2): 251-7, 2000 Oct 31.
Article in English | MEDLINE | ID: mdl-11080591

ABSTRACT

Nucleoside diphosphate kinase (NDPK) is a highly conserved, multifunctional enzyme. Its originally described function is the phosphorylation of nucleoside diphosphates to the corresponding triphosphates, using ATP as the phosphate donor and a high-energy phosphorylated histidine residue as the reaction intermediate. More recently, a host of additional functions of NDPK have been discovered. Some of these correlate with the capacity of NDPK to transphosphorylate other proteins, in a manner reminiscent of bacterial two-component systems. Other functions may be mediated by direct DNA-binding of NDPK. This study describes the identification of NDPK from the parasitic protozoon Trypanosoma brucei. The genome of this major disease agent contains a single gene for NDPK. The predicted amino acid sequence of the trypanosomal enzyme is highly conserved with respect to all other species. The protein is constitutively expressed and is present in procyclic and in bloodstream forms. Immunofluorescence and immuno-electron microscopy demonstrate that trypanosomal NDPK (TbNDPK) is predominantly localized in the cell nucleus. Histidine phosphorylation of TbNDPK is essentially resistant to the experimental compound LY266500, a potent inhibitor of histidine phosphorylation of trypanosomal succinyl coenzyme A synthase.


Subject(s)
Nucleoside-Diphosphate Kinase/genetics , Trypanosoma brucei brucei/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Histidine/metabolism , Microscopy, Electron , Molecular Sequence Data , Nuclear Proteins/metabolism , Nucleoside-Diphosphate Kinase/metabolism , Phosphorylation/drug effects , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Thiazoles/pharmacology , Trypanosoma brucei brucei/cytology , Trypanosoma brucei brucei/ultrastructure
2.
Mol Biochem Parasitol ; 100(1): 43-52, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10376992

ABSTRACT

The insect form of Trypanosoma brucei depends on respiration for its energy requirements. It contains a fully functional mitochondrion with a complete citric acid cycle. Most of its enyzmes have been characterized to date. The current study presents the characterization of the histidine phosphorylation activity of one of the few remaining enzymes, succinyl CoA synthetase. The trypanosomal enyzme was identified by partial purification, followed by direct protein sequencing. It is rapidly phosphorylated, presumably through auto-phosphorylation, using either ATP or GTP as phosphate donors. The phosphorylation occurs exclusively on histidine residues. The histidine-bound phosphate can be donated to suitable phosphate acceptors in a rapid reaction. This phosphotransfer reaction is highly nucleotide selective, as only ADP, but none of the other nucleoside-diphosphates tested, can be used as a phosphate acceptor.


Subject(s)
Histidine/metabolism , Succinate-CoA Ligases/metabolism , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/growth & development , Adenosine Triphosphate/metabolism , Animals , Guanosine Triphosphate/metabolism , Kinetics , Mitochondria/enzymology , Phosphorylation , Succinate-CoA Ligases/chemistry , Succinate-CoA Ligases/isolation & purification
3.
Mol Biochem Parasitol ; 100(1): 53-9, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10376993

ABSTRACT

Recent drug screenings for new antibacterial drugs directed against histidine phospho-relay signalling pathways in bacteria have resulted in compounds which potently inhibit the histidine kinase activity of bacterial two-component systems. The present study demonstrates that one of these compounds, LY266500, is also a potent inhibitor of histidine phosphorylation in the unicellular eukaryotic parasite Trypanosoma brucei, both in vitro and in whole cells. In vitro, it inhibits histidine phosphorylation of mitochondrial succinyl CoA synthetase. LY26650 does not interfere with the phosphotransfer from the histidine-phosphorylated protein to ADP. In standardized cell culture tests, LY266500 potently inhibits the proliferation of the human pathogens T. brucei rhodesiense and Leishmania donovani. Since the inhibitory activity in vivo is life-cycle stage specific and correlates well with the mitochondrial activity in the different stages, the effect of LY266500 is most likely due to its specific inhibition of the mitochondrial succinyl CoA synthetase.


Subject(s)
Enzyme Inhibitors/pharmacology , Protein Kinases , Succinate-CoA Ligases/antagonists & inhibitors , Thiazoles/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/enzymology , Animals , Histidine/metabolism , Histidine Kinase , Humans , Leishmania donovani/drug effects , Mitochondria/enzymology , Phosphorylation , Protein Kinase Inhibitors , Signal Transduction/drug effects , Succinate-CoA Ligases/metabolism , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/growth & development , Trypanosoma brucei rhodesiense/drug effects
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