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

摘要

The metal-dependent deacetylase UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine dea-ncetylase (LpxC) catalyzes the first committed step in lipid A biosynthesis, the hydrolysis of UDP-3-O-nmyristoyl-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate. Consequently, LpxC isna target for the development of antibiotics, nearly all of which coordinate the active site metal ion. Here wenexamine the ability of Fen2þ to serve as a cofactor for wild-type Escherichia coli LpxC and a mutant enzymen(EcC63A), in which one of the ligands for the inhibitory metal binding site has been removed. LpxC exhibitsnhigher activity (6-8-fold) with a single bound Fen2þ as the cofactor compared to Zn2þ-LpxC; bothnmetalloenzymes have a bell-shaped dependence on pH with similar pKa values, indicating that at least twonionizations are important for maximal activity. X-ray absorption spectroscopy experiments suggest that thencatalytic metal ion bound to Fen2þ-EcLpxC is five-coordinate, suggesting that catalytic activity may correlatenwith coordination number. Furthermore, the ligand affinity of Fen2þ-LpxC compared to the Zn2þ enzyme isnaltered by up to 6-fold. In contrast to Zn2þ-LpxC, the activity of Fen2þ-LpxC is redox-sensitive, and a time-ndependent decrease in activity is observed under aerobic conditions. The LpxC activity of crude E. coli cellnlysates is also aerobically sensitive, consistent with the presence of Fen2þ-LpxC. These data indicate thatnEcLpxC can use either Fen2þ or Zn2þ to activate catalysis in vitro and possibly in vivo, which may allow LpxCnto function in E. coli grown under different environmental conditions.