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Title: The characterisation of transketolase from Leishmania mexicana
Author: Veitch, Nicola Jean
ISNI:       0000 0001 3545 2304
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2002
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Transketolase is a key enzyme of the non-oxidative branch of the pentose phosphate pathway. The identification and characterisation of transketolase in Leishmania mexicana will aid in the understanding of the specific interactions of this enzyme and its products within this biochemical pathway. The pentose phosphate pathway may be a potential target for chemotherapy against Leishmania as it provides several key intermediates necessary for parasite survival within the host cell. In this study, the transketolase gene from L. mexicana was identified and cloned using a variety of molecular methodologies. The transketolase was shown to be similar to transketolases from other organisms, and contained two conserved motifs, a thiamine-pyrophosphate binding motif and a transketolase motif Analysis of the transketolase sequence revealed a carboxy-terminal peroxisome-targeting signal (PTS), suggestive of a glycosomal subcellular localisation. Subcellular fractionation of the promastigote Leishmania revealed that the protein was predominantly cytosolic, however a glycosomal localisation cannot be ruled out in other life cycle stages. Putative L. major and T. brucei transketolase protein sequences also contain a PTS at the C-terminal of the protein. The cloned transketolase gene was expressed as a recombinant protein using a bacterial system with the enzyme containing an N-terminal histidiae tag, allowing for efficient purification. Overexpression of the soluble transketolase protein occurred only at a low temperature (15°C). Characterisation of this recombinant transketolase is described. The specific activity of the L. mexicana transketolase was 1.65 U/mg and the Km for ribose 5-phosphate was 2.75 mM, which is similar to the Km values for this substrate measured in other species. Phylogenetic analysis of the L. mexicana protein sequence revealed an evolutionary relationship between the L. mexicana transketolase and the cyanobacterial and plant transketolases. This observation is consistent with the idea that several genes present in trypanosomatids are related to sequences of plant and cyanobacterial isoforms. The theory of the Kinetoplastida lineage losing a photo synthetic algal endosymbiont subsequent to plastid gene transfer into the nucleus of the parasite is discussed. Targeted gene replacement experiments were initiated to investigate the role transketolase plays within the Leishmania parasite. Constructs were prepared to replace transketolase with an antibiotic resistance marker via homologous recombination. The construction of the vectors and initial experiments are described.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral