Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site

UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria. Compounds ta rgeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli. A comparison of LpxC sequences from diverse bacteria identified 10 co nserved His, Asp, and Glu residues that might play catalytic roles. Each am ino acid was altered in both E. coli and Aquifex aiolicus LpxC and the cata lytic activities of the variants were determined. Three His and one Asp res idues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substituti ons at these positions. H79 and H238 likely coordinate zinc, the Zn2+ conte nt of the purified variant proteins is low and the specific activity is enh anced by the addition of Zn2+. The third side chain to coordinate zinc is l ikely either H265 or D246 and a fourth ligand is likely a water molecule, a s indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His( 2)AspH(2)O Zn2+-polyhedron in LpxC, The decreased zinc inhibition of LpxC m utants at E78 suggests that this side chain may coordinate a second, inhibi tory Zn2+ ion. Given the absence of any known Zn2+ binding motifs, the acti ve site of LpxC may have evolved differently than other well-studied zinc m etalloamidases, a feature that should aid in the design of safe antibiotics .