UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyz es the committed step in the biosynthesis of lipid A and is therefore a pot ential antibiotic target. Inhibition of this enzyme by hydroxamate compound s [Onishi, H. R.; Pelak, B. AI; Gerckens, L. S.; Silver, L. L.; Kahan, F. M ,; Chen, M, H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S,; Raetz, C IR, H. (1996) Science 274, 980-982] suggested the presence of a metal ion c ofactor. We have investigated the substrate specificity and metal dependenc e oft he deacetylase using spectroscopic and kinetic analyses. Comparison o f the steady-state kinetic parameters for the physiological substrate UDP-3 -O-(R-3-hydroxymyristoyl)GlcNAc and an alternative substrate, UDP-GlcNAc, d emonstrates that the ester-linked R-3-hydroxymyristoyl chain increases k(ca t)/K-M (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediamiuetetraacetic acid, completely inhibit LpxC activit y, implicating an essential metal ion. Plasma emission spectroscopy and col orimetric assays directly demonstrate that purified LpxC contains bound Zn2 +, This Zn2+ can be removed by incubation with DPA, causing a decrease in t he LpxC activity that can be restored by subsequent addition of Zn2+. Howev er, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no a dditional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu 2+. This is consistent with the profile of metals that substitute for catal ytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity max imally ata stoichiometry of 1:1. These data demonstrate that E. coli LpxC i s a metalloenzyme that requires bound Zn2+ for optimal activity.