Structural studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.
Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the essential steps in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Here, we report our studies on the secondary and quaternary structures of UNAM:L-Ala ligase from Escherichia coli. The molecular weight of the purified recombinant enzyme determined by electrospray ionization mass spectrometry agreed well with the molecular weight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monomeric and dimeric forms and that the dissociation constant of the dimer, Kd, was determined to be 1.1 +/- 0.4 microM at 37 degrees C and 0.58 +/- 0.30 microM at 4 degrees C. A very similar Kd value was also obtained at 37 degrees C by gel filtration chromatography. The secondary structure of the enzyme was characterized by circular dichroism spectroscopy. No change in the secondary structure was observed between the monomeric and dimeric forms of the enzyme. The activity assays at enzyme concentrations both below and above the determined Kd value lead to the conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization state.
Jin
H
H
Division of Macromolecular Structure and Analytical Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Emanuele
J J
JJ
Fairman
R
R
Robertson
J G
JG
Hail
M E
ME
Ho
H T
HT
Falk
P J
PJ
Villafranca
J J
JJ
D002500:Centrifugation, Isopycnic; D004926:Escherichia coli / Q000201:enzymology*; D013058:Mass Spectrometry; D008970:Molecular Weight; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase