The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases |
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Authors: | Emmanuel Mertens Uri S Ladror Jennifer A Lee Anya Miretsky Andrea Morris Catherine Rozario Robert G Kemp Miklós Müller |
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Institution: | (1) The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA, US;(2) Department of Biological Chemistry, Finch University of Health Sciences/The Chicago Medical School, North Chicago, IL 60064, USA, US |
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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 α- and β-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.
Received: 5 December 1997 / Accepted: 18 March 1998 |
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Keywords: | : Amitochondriate protist — Enzyme evolution — Glycolysis — Parabasala — Phosphofructokinase — PPi— Trichomonas |
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