Large-Scale Preparation, Purification, and Crystallization of UDP-N-Acetylmuramoyl-l-Alanine:d-Glutamate Ligase fromEscherichia coli☆

Abstract The UDP- N -acetylmuramoyl- l -alanine: d -glutamate ligase from Escherichia coli, an enzyme involved in the biosynthesis of the bacterial peptidoglycan monomer unit, was overproduced and purified to homogeneity on a large scale, yielding 4 mg of protein per liter of bacterial culture. Crystals of the complex with the substrate UDP-MurNAc- l -Ala were grown by the hanging drop method using ammonium sulfate as the precipitant. They are tetragonal with cell dimensions a = b = 65.5 A and c = 134.59 A, space group P4 1 or P4 3 , and contain one monomer of 46,842 Da in the asymmetric unit. In order to use the multiple-wavelength anomalous diffraction method for phasing, a selenomethionine derivative of the protein has also been overproduced, purified, and crystallized.