| Value |
103
Sec^-1
|
| Organism |
Bacteria Escherichia coli |
| Reference |
Smith KJ, Petit CM, Aubart K, Smyth M, McManus E, Jones J, Fosberry A, Lewis C, Lonetto M, Christensen SB. Structural variation and inhibitor binding in polypeptide deformylase from four different bacterial species. Protein Sci. 2003 Feb12 (2):349-60. Table - link PubMed ID12538898
|
| Method |
Cloning, expression and purification
PDF purified in the presence of nickel was used for all enzymatic and crystallographic studies. The pdf genes from S. aureus WCUH29, S. pneumoniae R6, E. coli K12, and H. influenzae Q1 were identified in proprietary sequence databases by BLAST homology searching. The genes were amplified by PCR from genomic DNA and cloned in pET plasmids (Novagen) and transformed into E. coli BL21(DE3). Enzyme assay
All the reactions were carried out in half-area 96-well microtiter plates (Corning) with a SpectraMax plate reader (Molecular Devices Corp.). PDF activity was measured at 25°C, using a continuous enzyme-linked assay developed by Lazennec and Meinnel (1997) with minor modifications. The catalytic properties of PDF enzymes from E. coli, H. influenzae, S. aureus, and S. pneumoniae toward the peptide substrate fMAS were assessed at pH 7.6 using a formate dehydrogenase coupling reaction. |
| Comments |
Newly synthesized polypeptide chains in prokaryotes carry a transiently formylated N terminus. The metalloprotease polypeptide deformylase (PDF) deformylates the N-formylmethionine group prior to removal of the N-terminal methionine by methionine aminopeptidase. Removal of the N-terminal methionine residue from polypeptide chains is thought to be important for biological activity and correct posttranslational modification and is strictly dependent on the removal of the N-formyl group by PDF. PDF has been cloned from a number of different species of bacteria and has been shown to be essential for bacterial cell viability |
| Entered by |
Uri M |
| ID |
104972 |