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-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.
Hernick
Marcy
M
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Gattis
Samuel G
SG
Penner-Hahn
James E
JE
Fierke
Carol A
CA
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007501:Iron / Q000494:pharmacology*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D056928:X-Ray Absorption Spectroscopy; D015032:Zinc / Q000378:metabolism
D003067:Coenzymes; D004791:Enzyme Inhibitors; D008024:Ligands; D007501:Iron; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase; D015032:Zinc; D010100:Oxygen