Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli.

Kohl, L.;Drmota, T.;Thi, C D;Callens, M.;Michels, Paulus;et.al.
(1996) Molecular and Biochemical Parasitology — Vol. 76, n° 1-2, p. 159-173 (1996)

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Authors
  • Kohl, L.
    Author
  • Drmota, T.
    Author
  • Thi, C DUCLouvain
    Author
  • Callens, M.
    Author
  • Opperdoes, FrederikUCLouvain
    Author
  • Michels, PaulusUCLouvain
    Author
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Abstract
A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.
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Kohl, L., Drmota, T., Thi, C. D., Callens, M., Van Beeumen, J., Opperdoes, F., & Michels, P. (1996). Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli. Molecular and Biochemical Parasitology, 76(1-2), 159-173. https://doi.org/10.1016/0166-6851(95)02556-1 (Original work published 1996)