Expression, characterization, and crystallization of the pyrophosphate-dependent phosphofructo-1-kinase of Borrelia burgdorferi.
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.
Deng
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Department of Microbiology and Immunology, Chicago Medical School, North Chicago, Illinois 60064, USA.
Roberts
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Wang
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Kemp
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RG
D000818:Animals; D015748:Borrelia burgdorferi Group / Q000201:enzymology*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D011756:Diphosphates / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016364:Reading Frames; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011756:Diphosphates; D011994:Recombinant Proteins; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase