Expression of recombinant diaminopimelate epimerase in Escherichia coli. Isolation and inhibition with an irreversible inhibitor.
Recombinant diaminopimelate epimerase is overproduced to give 1% of soluble protein when grown under the appropriate conditions in Escherichia coli. This compares with 0.02% of the constitutive level of wild-type enzyme. A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg). This has enabled sufficient amounts of enzyme both to compare with wild-type enzyme and to enable active site modification studies to be performed. Incubation of the enzyme with 2-(4-amino-4-carboxybutyl)-2-aziridine-carboxylic acid (AZIDAP), results in time-dependent irreversible inhibition. Tryptic digestion of the inactivated enzyme and peptide-mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide. Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequence of the dapF gene shows that Cys73 is labelled. Finally based on limited sequence similarities around this cysteine and active-site cysteines of proline racemase and 1-hydroxyproline 2-epimerase, together with mechanistic considerations, we propose that all three non-pyridoxal-phosphate-containing racemases/epimerases derive from a common evolutionary origin.
Higgins
W
W
Merrell Dow Research Institute, Strasbourg, France.
Tardif
C
C
Richaud
C
C
Krivanek
M A
MA
Cardin
A
A
D000591:Amino Acid Isomerases; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005798:Genes, Bacterial; D007535:Isomerases / Q000302:isolation & purification*; D008970:Molecular Weight; D010449:Peptide Mapping; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification
D011994:Recombinant Proteins; D007535:Isomerases; D019998:Racemases and Epimerases; D000591:Amino Acid Isomerases; C042032:diaminopimelate epimerase