UDP-3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase of Escherichia coli Is a Zinc Metalloenzyme

Abstract

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science274, 980−982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases k_cat/K_M (5 × 10⁶)-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn²⁺. This Zn²⁺ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn²⁺. However, 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 additional activity is observed upon the addition of Cd²⁺, Ca²⁺, Mg²⁺, or Cu²⁺. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co²⁺ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn²⁺ for optimal activity.