Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli.
Phosphofructokinase-2 (Pfk-2) belongs to the ribokinase family and catalyzes the ATP-dependent phosphorylation of fructose-6-phosphate, showing allosteric inhibition by a second ATP molecule. Several structures have been deposited on the PDB for this family of enzymes. A structure-based multiple sequence alignment of a non-redundant set of these proteins was used to infer phylogenetic relationships between family members with different specificities and to dissect between globally conserved positions and those common to phosphosugar kinases. We propose that phosphosugar kinases appeared early in the evolution of the ribokinase family. Also, we identified two conserved sequence motifs: the TR motif, not described previously, present in phosphosugar kinases but not in other members of the ribokinase family, and the globally conserved GXGD motif. Site-directed mutagenesis of R90 and D256 present in these motifs, indicate that R90 participates in the binding of the phosphorylated substrate and that D256 is involved in the phosphoryl transfer mechanism.
Cabrera
Ricardo
R
Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
Babul
Jorge
J
Guixé
Victoria
V
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D025481:Phosphofructokinase-2