Glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconolactonase (6PGL) from both T. brucei and Leishmania mexicana are present in both the cytosol and glycosomes (Duffieux et al., 2000). Moreover, both the T. brucei 6PGL and the Leishmania G6PDH carry a putative peroxisome-targeting signal (PTS): a specific C-terminal tripeptide (PTS1) or a nonapeptide close to the N-terminus (PTS2), respectively. This confirms that their respective genes encode truly glycosomal proteins. For G6PDH and 6PGL, we have previously proposed a plant affiliation and in the case of the G6PDH this was recently confirmed by Krepinsky et al. (2001) where the trypanosome G6PDH branches together with the G6PDHs from the plant cytosol.

Further details of the analysis

The Leishmania mexicana G6PDH was compared with the SwissProt release 39 / TrEMBL release 17 database indexed at European Bioinformatics Institute (EBI, Hinxton UK), containing 671,000 protein sequences, using the NCBI BLASTP program and the BLOSUM 62 matrix (http://www2.ebi.ac.uk/blastall/) . Click here to inspect the BLASTP output file. The best E value was obtained with the T. brucei G6PDH sequence Q9GRG7 . Out of 16 lesser scores in the BlastP output 13 were plant sequences while three were fungal G6PDH sequences.

The first 99 sequences from the blastp output were aligned using the "RunDBClustalW" option in the BLASTP output. The ClustalW alignment is availabe here for inspection. From this alignment incomplete sequences and positions with gaps were removed. This alignment in Phylip format containing 93 sequences and 332 positions was used for the calculation of pairwise distances between taxa.

The distance between the two trypanosomatid species was 29%, the distance between the trypanosomatids and the eukaryotes ranged from 40 to 48% and between trypanosomatids and the prokaryotes from 50-62%. This is suggestive of a eukaryotic origin of the trypanosomatid sequences. This was confirmed when the alignment was visually inspected. The trypanosomatid sequences shared many aspects with the other eukaryotic G6PDHs.

Using the neighbor-joining tree option of ClustalX a tree was prepared. Contrary to the result obtained with BLASTP, where the closest relatives to the trypanosomatid sequences were those of plants, the T. brucei sequence clustered with the fungal sequences, but bootstrap support for such a clustering was very weak. However, when some deeply branching taxa were removed form the dataset the trypanosomatid sequences became monophyletic with the plant cytosolic G6PDHs, but only with weak bootstrap support. Maximum parsimony, placed the trypanosomatid sequences together with those of the fungi.

Likelihood mapping as implemented in PUZZLE version 4.0.1. indicated the presence in the dataset of a strong phylogenetic signal (only 2.3% star-like quartets while 95% of the quartet trees were well-resolved).

Four-cluster likelihood mapping was carried out to test the support for an association of the L. mexicana and T. brucei G6PDHs (group a) with the plant chloroplast (group b), as opposed to the fungal G6PDHs (groups c) and the other eukaryotic G6PDHs (group d). The result showed that 75 % of quartet trees supported the clustering of the trypanosomatids with the fungi, whereas only 19 % favoured their grouping with the plant cytosolic G6PDHs.

Conclusion

The trypanosomatid G6PDHs are clearly eukaryotic. Although the dataset contains a strong phylogenetic signal, it is not possible to extract clear information about the affiliation of the trypanosomatid sequences. This is based on BlastP searches and on phylogenetic inference using distance matrix and neighbor-joining methods suggest that the trypanosomatid G6PDHs are more related to the cytosolic G6PDHs from plants, but bootstrap support is not robust. Maximum parsimony and likelihood mapping suggest an affiliation with the fungi rather than with the plants.