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-P...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 1999-11, Vol.371 (2), p.326-331
Main Authors: Deng, Zhihong, Roberts, David, Wang, Xiaojun, Kemp, Robert G
Format: Article
Language:English
Subjects:
Animals
Borrelia burgdorferi
Borrelia burgdorferi Group - enzymology
cloning and expression
Cloning, Molecular
Crystallography, X-Ray
Diphosphates - metabolism
Entamoeba histolytica - enzymology
Genes, Bacterial
Hydrogen-Ion Concentration
kinetic properties
Kinetics
phosphofructokinase
Phosphofructokinase-1 - chemistry
Phosphofructokinase-1 - genetics
Phosphofructokinase-1 - metabolism
pyrophosphate-dependent crystallization
Reading Frames
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
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Summary: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 Km values for fructose 6-P and PPi of 109 and 15 μM, 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.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.1999.1446