Activation of Escherichia coli UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine Deacetylase by Fe2+ Yields a More Efficient Enzyme with Altered Ligand Affinity

The metal-dependent deacetylase UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in lipid A biosynthesis, the hydrolysis of UDP-3-O-myristoylglucosamine to form UDP-3-O-myristoylglucosamine and acetate. Consequently, LpxC is a target for the development of antibiotics, nearly all of which coordinate the active site metal ion. Here we examine the ability of Fe to serve as a cofactor for wild-type Escherichia coli LpxC and a Mutant enzyme (EcC63A), in which one of the ligands for the inhibitory metal binding site has been removed. LpxC exhibits higher activity (6-8-fold) with a single bound Fe2+ as the cofactor compared to Zn2+-LpxC; both metalloenzymes have a bell-shaped dependence on pH with similar pK(a) Values, Indicating that at least two ionizations are important for maximal activity. X-ray absorption spectroscopy experiments Suggest that the catalytic metal ion bound to Fe2+-EcLpxC is five-coordinate, Suggesting that catalytic activity may correlate with coordination number. Furthermore, the ligand affinity of Fe2+-LpxC compared to the Zn2+ enzyme is altered by up to 6-fold. In contrast to Zn2+-LpxC, the activity of Fe2+-LpxC is redox-sensitive, and a time-dependent decrease in activity is observed Under aerobic conditions. The LpxC activity of crude E. coli cell lysates is also aerobically sensitive, consistent with the presence of Fe2+-LpxC. These data indicate that EcLpxC can use either Fe2+ or Zn2+ to activate catalysis in vitro and possibly in vivo, which may allow LpxC to function in E. coli grown under differentiation environmental conditions.