The pyrophosphate-dependent phosphofructokinase of the protist, Trichomonas vaginalis, and the evolutionary relationships of protist phosphofructokinases.

The pyrophosphate-dependent phosphofructokinase (PPi-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5' half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PPi-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PPi-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PPi-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the alpha- and beta-subunits of plant PPi-PFKs. The third group ("X") containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group ("Y") comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PPi-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers.

A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote, Trichomonas vaginalis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD+ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica--Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium.