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

The pyrophosphate-dependent phosphofructokinase (PPi-PFK) of the amitochond riate protist Trichomonas vaginalis has been purified. The enzyme is a homo tetramer of about 50 kDa subunits and is not subject to allosteric regulati on. The protein was fragmented and a number of peptides were sequenced. Bas ed on this information a PCR product was obtained from T. vaginalis gDNA an d used to isolate corresponding cDNA and gDNA clones. Southern analysis ind icated the presence of five genes. One open reading frame (ORF) was complet ely sequenced and for two others the 5' half of the gene was determined. Th e sequences were highly similar. The complete ORF corresponded to a polypep tide of about 46 kDa. All the peptide sequences obtained were present in th e derived sequences. The complete ORF was highly similar to that of other P FKs, 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 compar ison and phylogenetic reconstruction revealed a significant divergence amon g PPi-PFKs and related enzymes, which can be assigned to at least four dist antly related groups, three of which contain enzymes of protists. The separ ation of these groups is supported with a high percentage of bootstrap prop ortions. The short T. vaginalis PFK shares a most recent common ancestor wi th the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histo lytica 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 Trypa nosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorfe ri. The fourth group ("Y") comprises cyanobacterial and high-G + C, Gram-po sitive 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 phy logenetic analysis of which confirmed relationships established earlier. Th ese findings indicate a complex history of a key step of glycolysis in prot ists with several early gene duplications and possible horizontal gene tran sfers.