ABSTRACT
Cells of the actinomycete Amycolatopsis methanolica grown on glucose possess only a single, exclusively PP(i)-dependent phosphofructokinase (PP(i)-PFK) (A. M. C. R. Alves, G. J. W. Euverink, H. J. Hektor, J. van der Vlag, W. Vrijbloed, D.H.A. Hondmann, J. Visser, and L. Dijkhuizen, J. Bacteriol. 176:6827-6835, 1994). When this methylotrophic bacterium is grown on one-carbon (C(1)) compounds (e.g., methanol), an ATP-dependent phosphofructokinase (ATP-PFK) activity is specifically induced, completely replacing the PP(i)-PFK. The two A. methanolica PFK isoenzymes have very distinct functions, namely, in the metabolism of C(6) and C(1) carbon substrates. This is the first report providing biochemical evidence for the presence and physiological roles of PP(i)-PFK and ATP-PFK isoenzymes in a bacterium. The novel ATP-PFK enzyme was purified to homogeneity and characterized in detail at the biochemical and molecular levels. The A. methanolica ATP-PFK and PP(i)-PFK proteins possess a low level of amino acid sequence similarity (24%), clearly showing that the two proteins are not the result of a gene duplication event. PP(i)-PFK is closely related to other (putative) actinomycete PFK enzymes. Surprisingly, the A. methanolica ATP-PFK is most similar to ATP-PFK from the protozoon Trypanosoma brucei and PP(i)-PFK proteins from the bacteria Borrelia burgdorferi and Treponema pallidum, both spirochetes, very distinct from actinomycetes. The data thus suggest that A. methanolica obtained the ATP-PFK-encoding gene via a lateral gene transfer event.
Subject(s)
Actinomycetales/enzymology , Diphosphates/metabolism , Phosphofructokinase-1/metabolism , Phosphotransferases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Carbon/metabolism , Cloning, Molecular , Culture Media , Glucose/metabolism , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Methanol/metabolism , Models, Biological , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Phosphofructokinase-1/classification , Phosphofructokinase-1/isolation & purification , Phosphorus Isotopes , Phylogeny , Sequence Analysis, DNA , Sequence Analysis, Protein , Sequence Homology, Amino AcidABSTRACT
The pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) from Giardia lamblia has been purified to homogeneity using two methods. The purified enzyme is shown to be homogenous by its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single peak on reverse-phase HPLC. The purified enzyme is a monomeric protein of Mr 64 kDa as determined by SDS-PAGE and native PAGE. The enzyme fractionated as a 67-kDa protein by HPLC gel filtration. The pH optima in either the glycolytic or the gluconeogenic direction is near neutral, which is unlike any of the protozoan or bacterial enzymes. The enzyme displayed typical Michaelis-Menten kinetics. The Km values for pyrophosphate and fructose 6-phosphate were 0.039 +/- 0.005 and 0.25 +/- 0.0196 mM, respectively. The initial velocity for the reverse reaction was also found to be hyperbolic. Different nucleoside triphosphates, including ATP, could not substitute for pyrophosphate as the phosphoryl donor. Similar to the pyrophosphate-dependent enzyme from other anaerobic protozoans and bacteria, the Giardial enzyme was not activated by fructose 2,6-bisphosphate, although this metabolite is a competitive inhibitor with respect to fructose 1,6-bisphosphate in the reverse reaction. The pH optima and the molecular weight properties of the Giardial enzyme are different from those of the two classes of the pyrophosphate-dependent phosphofructokinases.
