This page gives access to the supplementary material belonging to the paper:

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei must have evolved from an ancestral pyrophosphate-dependent enzyme

Paul A.M. Michels, Nathalie Chevalier, Fred R. Opperdoes, Mark H. Rider and Daniel J. Rigden

Creation of a multiple alignment
A multiple alignment containing 43 ATP- and PPi-dependent PFK sequences was created using the program ClustalW. The alignment was adjusted manually by using information on the secondary structure of the E. coli ATP-PFK. From this alignment a sub-collection of relevant sequences was made as shown in Fig. 3 in the paper.

Phylogenetic analyses.
From the combined alignment of all available ATP-dependent and PPi-dependent PFKs, those regions that contained major insertions or deletions, or those where the alignment was ambiguous, were removed). The following columns in the alignment were retained: 271,296:310,351:375,388:392,446:450,479:498,505:509,522:602,642. The resulting condensed alignment file in Phylip format containing the remaining 228 sites was used in phylogenetic analyses with either the neighbor-joining, maximum parsimony, or the maximum likelihood method. All three methods gave highly similar results and the neighbor-joining and maximum likelihood trees are available. The clustering into separate clades of the eukaryotic ATP-PFKs (N- and C-terminal domains), the bacterial ATP-dependent PFKs, as well as a all PPi-related PFKs, was supported by bootstrapping analysis for both methods applied. Slight differences between the methods were observed. Maximum likelihoodwas not able to resolve the relative branching order within some of the clades such as the bacterial ATP-PFK clade. Neither did it resolve the exact order of the separation between bacterial and eukaryotic ATP-PFKs and the gene duplication in the latter. In the neighbor-joining method Propionobacterium. freudenreichii branched off before the separation of N. fowleri, E. histolitica and T. brucei, whereas in the ML method it grouped together with the three protists sequences. Under maximum parsimony the three protist PFKs formed one clade (not shown). Also in the ML two the helminths, S. mansoni and H. contortus, were monophyletic, while with neighbor joining they behaved a paraphyletic taxa. A distance matrix was prepared after conversion of the alignment file in genbbank format to a pir/nbrf format followed by importation into ClustalW. The Uncorrected distance matrix was prepared with the settings "correction for multiple mutations" = off and "exclusion of regions with gaps" = off.

Supplementary materials available:

• T. brucei PFK nucleic-acid sequence accession code: AF008186
• T. brucei PFK amino-acid sequence
• Multiple sequence alignments:
  • multiple alignment of all 43 PFK sequences in genbank format
  • multiple alignment of all 43 PFK sequences in msf format
  • multiple alignment of all 43 PFK sequences in jpeg format
  • multiple alignment of all 43 PFK sequences in postscript format
  • multiple alignment of selected sequences in jpeg format
  • alignment of the PFK 228 used in the phylogenetic analyses
• Phylogenetic trees:
  • neighbor-joining tree in jpeg format
  • maximum likelihood tree in jpeg format
• Accession numbers of the PFK sequences used in the study
• Uncorrected distance matrix

Access the directory with all avaialable files