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) Science 274, 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 kcat/KM (5 x 10(6))-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.

Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis

“…We suspected that the anomalous scattering of a single zinc ion bound to a polypeptide chain of 271 residues would be insufficient for the calculation of MAD phases. Therefore, we exploited the fact that LpxC, like many zinc proteases, is inhibited by excess zinc (17). We expected to find that the preparation of LpxC crystals in the presence of millimolar concentrations of Zn 2ϩ would lead to the binding of additional zinc ions, which in turn would facilitate MAD phasing.…”

“…A. aeolicus LpxC was stripped of all metal ions by dialysis against 1.0 mM EDTA in 25 mM Hepes (pH 7.0)͞0.1 M NaCl at room temperature for Ն4 h. The EDTA was then removed by extensive dialysis against EDTA-free buffer and the enzyme was reconstituted to a 1:1 Zn 2ϩ :LpxC ratio by the addition of ZnSO 4 . A colorimetric assay employing 4-(2-pyridylazo)-resorcinol (PAR) was used to determine Zn 2ϩ concentrations (17) and verify the preparation of apo and 1:1-reconstituted LpxC. The calorimeter cell contained either Ϸ40 or Ϸ60 M enzyme, and the syringe contained 250 or 400 M aliphatic compound.…”

“…1) and is essential for bacterial growth and virulence (16). Although LpxC is a zinc metalloenzyme (17), the lack of characteristic zinc-binding motifs (18) in its amino acid sequence suggests that this enzyme represents a class of zinc-dependent deacetylases (17). Recent mutagenesis studies (19) with LpxC enzymes from Escherichia coli and Aquifex aeolicus indicate residues important for catalysis and zinc binding.…”

Structure of the Bacterial Deacetylase LpxC Bound to the Nucleotide Reaction Product Reveals Mechanisms of Oxyanion Stabilization and Proton Transfer

“…Protein Purification and Crystallization-E. coli LpxC was cloned and purified as described previously (31) with the exception of a C125S mutation. Protein was concentrated in 20 mM Hepes, pH 7.0, 50 mM NaCl, and 0.5 mM zinc sulfate to 12 mg/ml (0.35 mM), as determined by absorbance at 280 nm using a calculated extinction coefficient of 22 (36).…”

Binding of Uridine 5‘-Diphosphate in the “Basic Patch” of the Zinc Deacetylase LpxC and Implications for Substrate Binding,

“…The C193A/ΔD284-L294 variant of LpxC from A. aeolicus (henceforth "LpxC") was overexpressed in E. coli and purified as described (19,28) and crystallized using previously reported conditions (23). To prepare the UDP complex, crystals were gradually transferred to 100 mM HEPES (pH 8.0), 180 mM NaCl, 14-16% PEG 3350, 0.5 mM ZnSO 4 , 2 mM MgCl 2 , and 30 mM UDP, and soaked for 16 hours.…”