Expression, characterization, and crystallization of the pyrophosphate-dependent phosphofructo-1-kinase of Borrelia burgdorferi

Arch Biochem Biophys. 1999 Nov 15;371(2):326-31. doi: 10.1006/abbi.1999.1446.

Abstract

The two genes for the putative pyrophosphate-dependent phosphofructokinases (PPi-PFKs) of Borrelia burgdorferi were cloned by PCR and expressed in Escherichia coli, and their protein products were purified to near homogeneity. The larger of the two gene products, a 62-kDa protein, is an active PPi-PFK and exists in solution as a dimer. It has apparent K(m) values for fructose 6-P and PPi of 109 and 15 microM, respectively, and a pH optimum of 6.4 to 7.2. The 62-kDa protein was crystallized and subjected to preliminary diffraction analysis. The smaller gene product, a 48-kDa protein, exists in solution as a higher polymer and shows no ATP- or PPi-dependent activity, despite having a secondary structure as estimated by circular dichroism that is not significantly different from that of other PFKs.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Borrelia burgdorferi Group / enzymology*
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Diphosphates / metabolism
  • Entamoeba histolytica / enzymology
  • Genes, Bacterial
  • Hydrogen-Ion Concentration
  • Kinetics
  • Phosphofructokinase-1 / chemistry*
  • Phosphofructokinase-1 / genetics
  • Phosphofructokinase-1 / metabolism
  • Reading Frames
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Diphosphates
  • Recombinant Proteins
  • Phosphofructokinase-1
  • 1-phosphofructokinase