Alternative oxidase

Another typical plant-like enzyme found in the African trypanosomes and in Phytomonas spp. and for which its origin was never understood, is a very active cyanide-insensitive terminal oxidase which makes part of the trypanosomes' mitochondrial respiratory chain and which is inhibited by salicylhydroxamic acid, similar to the alternative oxidase (AOX) of plants (Chaudhuri and Hill., 1998). Its sequence and that from plants and fungi are available and they are all homologous proteins. Recently the sequence of the oxidase of the green alga Chlamydomonas reinhardtii (O65000 and Q9FE26) also became available and this algal enzyme now turned out to be the closest relative of the enzyme from the trypanosome (AOX_TRYBB). Moreover, C. reinhardtii was reproducibly positioned basally of the trypanosome and plant sequences (Click here for a NJ tree).

Further details of the analysis

The T. brucei AOX 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.

Only the sequences with the best E values (4 e-46 to 6 e-32) were selected. These comprised in addition to the trypanosomatid sequence only plant and fungal sequences. No alternative oxidase has been reported for any other organism. Percentage identity with that of the T. brucei sequence, according to the BLAST algorithm, ranged from 44 to 39.

The sequences were aligned in Clustal. This alignment was freed of gaps and incomplete or duplicate sequences were removed resulting in an alignment of 32 sequences with 180 residues. The programme Clustal was used to calculate a table with uncorrected pairwise distances. Pairwise percentages of identity between overall sequences after removal of gaps ranged from 44 to 33, with the highest identity to Oryza sativa (rice).

The alignment in Phylip format containing 32 sequences and 180 sites was used for likelihood mapping as implemented in PUZZLE version 4.0.1. The result indicated that the dataset contained a good phylogenetic signal with only 4.7% of all quartets unresolved and with 91.2 % of fully resolved quartets.

Then a bootstrapped neigbor-joining tree was created in Clustal using the alignment in Phylip format above. The NJ tree (10 000 bootstrap samplings) is robust and the T. brucei sequence branches closely to the alga C.reinhardtii and forms a single clade with the plants with a bootstrap support of 98%. Also a maximum likelihood tree (10 000 puzzle steps) gave a similar topology with 95% of quartets supporting the clustering of T. brucei with the plants. The maximal parsimony tree (100 bootstrap samplings) gave the same topology and similar bootstrap support for the clustering of trypanosome with the plants.

Since the trypanosome and Chlamydomonas sequences are almost equidistant from both plants and fungi it is important to note that all three phylogenetic analyses placed the trypanosome robustly at the side of the plants with respect to the alga. The validity of such a placement was then tested by Four cluster likelihood mapping. This method showed that 97% of all quartets supported a branching that puts the trypanosomatids (a) together with the other plants (d) versus the alga (b) and the fungi (c). An association of the trypanosomatids (a) with the fungi (c) as was not supported at all (0%).

Conclusion

There is strong support for a clustering of the trypanosomatid AOX with that of plants. This is not only suggested by the BLASTP result, but also by the pairwise percentages of identity between the trypanosome sequence and the others, by three different phylogeny inference methods, as well as by likelihood mapping.