STRUCTURAL STUDIES OF ESCHERICHIA-COLI UDP-N-ACETYLMURAMATE-L-ALANINELIGASE

Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNA M:L-Ala Ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the ess ential steps in cell wall biosynthesis for both gram-positive and gram -negative bacteria. Here, we report our studies on the secondary and q uaternary structures of UNAM:L-Ala ligase from Escherichia coli. The m olecular weight of the purified recombinant enzyme determined by elect rospray ionization mass spectrometry agreed well with the molecular we ight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monome ric and dimeric forms and that the dissociation constant of the dimer, K-d, was determined to be 1.1 +/- 0.4 mu M at 37 degrees C and 0.58 /- 0.30 mu M at 4 degrees C. A very similar K-d value was also obtaine d at 37 degrees C by gel filtration chromatography. The secondary stru cture of the enzyme was characterized by circular dichroism spectrosco py. No change in the secondary structure was observed between the mono meric and dimeric forms of the enzyme. The activity assays at enzyme c oncentrations both below and above the determined K-d value lead to th e conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization s tate.