6-Phosphoglucononate dehydrogenase

The sequence of the third enzyme of the HMP pathway, the 6-phosphoglucononate dehydrogenase (6PGDH) has been cloned and sequenced (Barrett and Le Page, 1993). Phylogenetic analyses placed the T. brucei 6PGDH robustly in the prokaryotic clade, closely with the enzymes from the cyanobacteria Synechocystis and Synechococcus, with whom they were either monophyletic or paraphyletic. This is in agreement with recent reports by Krepinsky et al, 2001; and C. Greenblatt (personal communication) who cloned the Leishmania gene (unpublished). Click here for a phylogenetic tree

Further details of the analysis

The Leishmania enzyme was aligned with the other 6PGDH sequences.

The trypanosomatid 6PGDHs were compared with the SwissProt database indexed at European Bioinformatics Institute (EBI, Hinxton UK) on 28 July 2001 and having 99162 entries, 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 values (2e-87 to 2e-81) were all obtained with prokaryotic 6PGDHs. The first eukaryotic sequence was that of Schizosaccharomyces pombe (2e-81). The first 38 unambiguous 6PGDH sequences from the BLASTP output were aligned using the "RunDBClustalW" option. The ClustalW alignment is availabe here for inspection. From this alignment positions with gaps were removed and the alignment in Phylip format containing 39 sequences with 292 positions was used for the the calculation of pairwise distances. The Leishmania and T. brucei enzymes share 75% identical residues. The two trypanosomatid sequences were on average 38 to 44% identical with the other sequences available in the database. The T. brucei sequence shared between 43 and 37% identical residues with the other sequences.

Likelihood mapping as implemented in PUZZLE version 4.0.1. indicated the presence in the dataset of a strong phylogenetic signal (only 2.7% star-like quartets while 96 % of the quartet trees were well-resolved).

The alignment was also used for the creation of a bootstrapped neighbor-joining tree. The clustering of the Trypanosoma sequence with the prokaryotic sequences was robust, but its association with cyanobacteria as a paraphyletic group was only weakly supported by bootstrap values. The T. brucei has the longest branch in the tree. This may have an influence on the branching position of T. brucei 6PGDH due to the "long branch attraction artefact". Therefore, bootstrapped maximal parsimony and maximum likelihood trees were created as well. These trees had very similar topologies, always placing the Trypanosoma 6PGDH sequence robustly within the prokaryotic clade either monophyletic or paraphyletic with the cyanobacterial sequences.

Conclusion

Phylogenetic analyses place the T. brucei 6PGDH robustly in the prokaryotic clade, close to the enzymes from the cyanobacteria Synechocystis and Synechococcus.