Aromatic alpha-hydroxyacid
dehydrogenase
Cazzulo-Franke
et al. (2001) have recently described
in T. cruzi an aromatic alpha-hydroxyacid dehydrogenase (AHDH)
which carries a peroxisomal targeting signal of type 1 (PTS1) and
which has been localized to the glycosomes. This enzyme is involved
in the metabolism of aromatic amino acids. It is closely related to
the family of malate dehydrogenases (MDHs), but has apparently
changed its substrate specificity during evolution. Comparison of the
56 most similar MDH sequences showed that this T. cruzi AHDH
is most related to the MDHs from the plant cytosol. (Click
here
for a phylogenetic tree).
Further details of the analysis
A Trypanosoma cruzi aromatic-AHAD was compared with all
the sequences of the SwissProt
database indexed at European
Bioinformatics Institute (EBI, Hinxton UK), 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 scores are the trypanosomatid AHADH of T. cruzi
(Q9NJT2
and Q9NJT1)
and the cytosolic MDH of T. brucei ( AAK83037),
immediately followed by the plant cytosolic malate dehydrogenase
sequences, of which the scores ranged from E= 3e-89 to 3e-77 and
displaying 50 - 55% identity.
The best scoring sequences were selected for the creation of an alignment by Clustal. The alignment was freed of incomplete sequences, duplicates and gaps and the resulting alignment of 56 sequences with 284 sites was used to calculate a table with uncorrected pairwise distances. Pairwise percentages of identity of the trypanosomatid sequence with that of the plant cytosolic sequences ranged from 55-52%, while all other sequences shared between 51-36% identical residues.
A bootstrapped neigbor-joining tree (10 000 bootstrap samplings), a maximum likelihood (25 000 puzzling steps) and a bootstrapped maximum parsimony tree (100 bootstrap samplings) were created using the alignment in Phylip format. All three trees clustered the trypanosomatid sequence together with that of the plant cytosolic MDHs, in agreement with the BlastP result . However, bootstrap support for this clustering was weak and ranged from 62 (nj) to 44% (mp). Maximum likelihood gave a stronger support for such a clustering (88% puzzle quartet frequency).
Likelihood mapping indicated that the dataset contained a strong phylogenetic signal indeed. Only 1% of all quartets were unresolved while 97 % of the quartets were fully resolved. Using Four-cluster likelihood mapping the possibility that the T. cruzi sequence clustered with the other protist sequences (Trichomonas and Giardia) was tested. 56% of all quartets supported a branching of T. brucei AHAD (a) with the plant cytosolic sequences (c), while only 37% supported branching with the protist sequences (b). No support was found for a branching with the other eukaryotes (d).
Conclusion
The BlastP algorithm and the pairwise percentages of identity both
suggest a closer relationship of the trypanosome AHAD sequences with
that of the plant cytosolic MDHs. This is corroborated by
neighbor-joining and maximum likelihood trees, while the maximum
parsimony tree gives only weak support for such an association. While
four-cluster likelihood mapping also supports the clustering of the
trypanosomes with the plants, an association with the protist AHAD of
Trichomonas and Giardia cannot be excluded.