PURIFICATION AND CHARACTERIZATION OF THE D-ALANYL-D-ALANINE-ADDING ENZYME FROM ESCHERICHIA-COLI

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-γ-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 49000, and it has a turnover number of 784 min−1 for ATP-driven amide bond formation. Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-γ-D-Glu-mejo-2,6-diaminopimelate and D-trifluoroalanine proved that the preceding 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-iV-acetylmuramyl-L-Ala-γ-D-Glu-meso-2,6-diaminopimelyl-D-trifluoroAla-D-trifluoroAla. © 1990, American Chemical Society. All rights reserved.