PMID:20136146
| Citation |
Hernick, M, Gattis, SG, Penner-Hahn, JE and Fierke, CA (2010) Activation of Escherichia coli UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase by Fe2+ yields a more efficient enzyme with altered ligand affinity. Biochemistry 49:2246-55 |
|---|---|
| Abstract |
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-myristoyl-N-acetylglucosamine 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(2+) 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 Fe(2+) as the cofactor compared to Zn(2+)-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 Fe(2+)-EcLpxC is five-coordinate, suggesting that catalytic activity may correlate with coordination number. Furthermore, the ligand affinity of Fe(2+)-LpxC compared to the Zn(2+) enzyme is altered by up to 6-fold. In contrast to Zn(2+)-LpxC, the activity of Fe(2+)-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 Fe(2+)-LpxC. These data indicate that EcLpxC can use either Fe(2+) or Zn(2+) to activate catalysis in vitro and possibly in vivo, which may allow LpxC to function in E. coli grown under different environmental conditions. |
| Links |
PubMed PMC2884013 Online version:10.1021/bi902066t |
| Keywords |
Amidohydrolases/antagonists & inhibitors; Amidohydrolases/chemistry; Amidohydrolases/genetics; Amidohydrolases/metabolism; Animals; Biocatalysis; Catalytic Domain; Coenzymes/metabolism; Enzyme Activation/drug effects; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Iron/pharmacology; Ligands; Models, Molecular; Mutation; Oxygen/metabolism; Protein Binding; X-Ray Absorption Spectroscopy; Zinc/metabolism |
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