The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

Abstract

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.

The Two Phosphofructokinase Gene Products of Entamoeba histolytica

“…Phylogenetic studies of the sequences of PFKs place the two E. histolytica PFKs in a large group of proteins, most of which have been described as PP i -PFKs that are distinct from the typical ATP-PFKs such as those found in E. coli as well as all mesozoans. The 60-kDa enzyme falls into a monophyletic subgroup that contains a number of other well characterized PP i -PFKs including those of plants (22,23). On the other hand, the 48-kDa PFK sequence from E. histolytica falls into a monophyletic subgroup within the PP i -PFK group that also contains Treponema pallidum and Borrelia burgdorferi and the peroxisomal ATP-PFK of Trypanosoma brucei (22,23).…”

“…The 60-kDa enzyme falls into a monophyletic subgroup that contains a number of other well characterized PP i -PFKs including those of plants (22,23). On the other hand, the 48-kDa PFK sequence from E. histolytica falls into a monophyletic subgroup within the PP i -PFK group that also contains Treponema pallidum and Borrelia burgdorferi and the peroxisomal ATP-PFK of Trypanosoma brucei (22,23). Of the other three members of the group, the T. pallidum gene product has not been characterized and preliminary studies of the B. burgdorferi product have not found either ATP-or PP i -PFK activity (24).…”

Presence of Prokaryotic and Eukaryotic Species in All Subgroups of the PP i -Dependent Group II Phosphofructokinase Protein Family

“…Based primarily on sequence characteristics, type A PFKs are currently assigned to three major groups (groups I, II, and III) (22). Group II can be further subdivided into four subgroups, which appear as robust clades in phylogenetic reconstructions (we are using the tentative nomenclature proposed for clades in group II [18]). Closely related organisms may contain close homologs of PFK which use different phosphoryl donors, indicating that enzyme specificity can change relatively easily (2), a conclusion recently confirmed experimentally (7).…”

“…The order of separation of lineages within these clades has not been resolved, however. While this subdivision is based only on the easily aligned amino-terminal parts of the sequences, a global comparison of the complete sequences and an analysis of the lengths and positions of insertions and deletions result in an identical subdivision (18).…”

“…So far, no archaebacterial sequences were noted in PFK group II. While the products of a number of sequences analyzed remain to be studied, available information indicates that the long, short, and P (new designation) clades correspond to PP i -specific enzymes (10,18) and that clade X contains ATP-specific PFKs (8,19).…”