Cloning, sequencing, and expression of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.
Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii is a non-allosteric enzyme with properties dissimilar to those of other described phosphofructokinases. The enzyme was cloned into pBluescript, sequenced, and expressed in Escherichia coli at levels 15 times higher than those observed in Propionibacterium. The gene consists of 1215 bases which code for a protein of 404 amino acids and a mass of 43,243 daltons. High G + C in the codon usage (66%) of the gene is consistent with the classification of Propionibacterium in the High-G + C subdivision of the Gram-positive bacteria. While showing no sequence identity to the non-allosteric ATP-dependent phosphofructokinase of E. coli, alignments of the amino acid sequence with other PFKs reveal degrees of identities among the amino halves of the proteins, from 26% between the Propionibacterium and potato PPi-PFKs, and 29% between Propionibacterium and E. coli ATP-PFKs. These levels of identities indicate that the amino halves of these proteins are homologous. Identities between the carboxyl half of Propionibacterium PFK and carboxyl halves of other sequences are below 20%, suggesting that the carboxyl half is not homologous. Despite the poor conservation, most of the residues that take part in the binding of fructose-6-P or Mg-PPi could be readily identified by analogy to the structure of the E. coli PFK. Both the fructose-6-P and ATP-binding sites are conserved, indicating that PPi binds to the homologous site of the E. coli ATP-binding site.
Ladror
U S
US
Department of Biological Chemistry, University of Health Sciences, Chicago Medical School, Illinois 60064.
Gollapudi
L
L
Tripathi
R L
RL
Latshaw
S P
SP
Kemp
R G
RG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D011424:Propionibacterium / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
D004269:DNA, Bacterial; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D010732:Phosphofructokinase-1; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase