Phase determination of the UDP-N-acetylmuramic acid :L-alanine ligase (MurC) crystal from Mycobacterium bovis

Bacterial peptidoglycan is necessary for bacterial survival against environmental osmotic pressure and is a relevant target for development of anti-bacterial drugs. Formation of a covalent bond between a carbohydrate and an amino acid is a key chemical process for peptidoglycan bio-synthesis. UDP-N-acetylmuramic acid (UDPMurNAc):L-alanine ligase (MurC) is an ATP-dependent amide bond ligase to form an UDP-MurNAc-L-alanine required in bacterial peptidoglycan. To provide a structural background for development of tuberculosis-specific antibiotics, Mycobacterium bovis MurC (MbMurC), which has sequence identity of 100% to M. tuberculosis, was cloned and expressed. The purified protein was crystallized from the precipitant of 0.1 M HEPES (pH 7.0), 0.2 M NaCl, 24% (w/v) polyethylene glycol 1.5K, and 10% (v/v) 2-methyl-2,4-pentanediol. Diffraction data were collected to 2.3 Å resolution. The crystal belonged to the primitive monoclinic space P2 1 with unit-cell parameters a = 65.30 Å, b = 76.70 Å, c =103.96 Å, α = γ = 90°, and β = 106.0°. The spatial positions of the two protein molecules in the asymmetric unit were determined by molecular replacement using the sequentially related Yersinia pestis MurC structure.