title: Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase from Escherichia coli.
abstract: The UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase was over-produced and purified from two plasmid-harbouring strains of Escherichia coli. The first strain, E. coli JM83(pHE5), gave a 15-fold over-production relative to parental strain. The enzyme could be partially purified (8.8-fold) by ion-exchange chromatography. With the second strain, E. coli JM83(pMLD25), a very strong over-production was obtained, since the enzyme represented about 20% of the cytoplasmic proteins. Purification yielded 77% protein homogeneity. However, the enzymatic activity, which was very unstable, was lost during the purification procedure. Several properties of the enzyme were studied. The enzyme gave maximal activity around pH 8. The isoelectric point was 5.2. The activity was increased by potassium phosphate. Reverse and exchange reactions could be catalysed. The N-terminal sequence of the protein was determined and correlated with the nucleotide sequence of the murE gene. The actual initiation codon was assigned.
authors: Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.
authors: C
authors: C
authors: Michaud
authors: D
authors: D
authors: Mengin-Lecreulx
authors: J
authors: J
authors: van Heijenoort
authors: D
authors: D
authors: Blanot
mesh_terms: D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010453:Peptide Synthases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D012492:Salts
chemical_list: D012492:Salts; D010453:Peptide Synthases; C049145:UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase