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.
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.
Kohl
L
L
Research Unit for Tropical Diseases, Catholic University of Louvain, Brussels, Belgium.
Drmota
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Thi
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Callens
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Van Beeumen
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Opperdoes
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Michels
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D015870:Gene Expression; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010802:Phylogeny; D017385:Sequence Homology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D009243:NAD; D005993:Glycerolphosphate Dehydrogenase; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+)