Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli

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Abstract
The Escherichia coli D-alanyl-D-alanine-adding enzyme, which catalyzes the final cytoplasmic step in the biosynthesis of the bacterial peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-.gamma.-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala, has been purified to homogeneity from an E. coli strain that harbors a recombinant plasmid bearing the structural gene for this enzyme, murF. The enzyme is a monomer of molecular weight 49 000, and it has a turnover number of 784 min-1 of ATP-driven amide bond formation. Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-.gamma.-D-Glu-meso-2,6-diaminopimelate and D-trifluoroalanie proved that the preceeding enzyme in the D-alanine branch pathway, D-alanine:D-alanine ligase (ADP), is capable of synthesizing fluorinated dipeptides, which the D-Ala-D-Ala-adding enzyme can then incorporate to form UDP-N-acetylmuramyl-L-Ala-.gamma.-D-Glu-meso-2,6-di-aminopimelyl-D-trifluoroAla-D-trifluoroAla.