Active site metal ion in UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) switches between Fe(II) and Zn(II) depending on cellular conditions

Abstract

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate in Gram-negative bacteria. This second, and committed, step in lipid A biosynthesis is a target for antibiotic development. LpxC was previously identified as a mononuclear Zn(II) metalloenzyme; however, LpxC is 6-8-fold more active with the oxygen-sensitive Fe(II) cofactor (Hernick, M., Gattis, S. G., Penner-Hahn, J. E., and Fierke, C. A. (2010) Biochemistry 49, 2246-2255). To analyze the native metal cofactor bound to LpxC, we developed a pulldown method to rapidly purify tagged LpxC under anaerobic conditions. The metal bound to LpxC purified from Escherichia coli grown in minimal medium is mainly Fe(II). However, the ratio of iron/zinc bound to LpxC varies with the metal content of the medium. Furthermore, the iron/zinc ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared with Fe(II) with K(D) values of 60 ± 20 pM and 110 ± 40 nM, respectively. However, in vivo concentrations of readily exchangeable iron are significantly higher than zinc, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between iron and zinc in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.

FIGURE 1.

A, role of LpxC in lipid A biosynthesis. B, active site of ZnLpxC derived from Protein Data Bank code 1P42 (49). C, proposed chemical mechanism of LpxC catalysis (16).

FIGURE 2.

Metal content of EcC63A LpxC-ZZ isolated from E. coli. A, EcC63A LpxC-ZZ was expressed in BL21(DE3) pEcC63ALpxC-ZZ E. coli grown in minimal medium with and without iron or zinc supplementation (5–20 μm) and induced by addition of either 1 mm isopropyl β-d-thiogalactopyranoside (closed circles) or 0.1 mm isopropyl β-d-thiogalactopyranoside (open circles). The protein was purified using an IgG pulldown under anaerobic conditions (see “Materials and Methods”). The iron/zinc ratio of metals bound to LpxC-ZZ (circles) and in the cleared lysate was analyzed by ICP-MS. The iron/zinc ratio in natively expressed LpxC determined by activity (triangles), as described in the legend of Fig. 3, mirror the pulldown data. B, as controls, purified FeLpxC-ZZ (closed circles) or ZnLpxC-ZZ (open circles) was added to lysates of BL21(DE3) pEcC63ALpxC E. coli cells grown in minimal medium with and without iron or zinc supplementation (5–20 μm) and repurified by IgG-pulldown, as described under “Materials and Methods.” LpxC-ZZ retains the majority of the original metal ion and does not readily equilibrate with metal ions in the lysate. Trendline reflects data in A.

FIGURE 3.

Native LpxC activity varies with metal supplementation in the growth medium. E. coli BL21(DE3) cells (without LpxC expression plasmid) were grown in minimal medium with and without 20 μm metal supplementation, lysed, and assayed for deacetylase activity as described under “Materials and Methods” either anaerobically (black bars) or after exposure to room oxygen for 2.5 h (gray bars).

FIGURE 4.

C63A-LpxC readily equilibrates with metal ions in vitro and has a significantly higher affinity for Zn(II) than Fe(II). A, affinity of C63A-LpxC for Zn(II) is 1800-fold higher than Fe(II). Apo-C63A-LpxC (untagged) was equilibrated with buffered metal solutions (5 mm Mops, 1 mm NTA, pH 7, as described under “Materials and Methods”). Bound zinc (circles) was analyzed by ultrafiltration and ICP-MS analysis, (K_D^Zn) and bound iron (diamonds) was analyzed by enhancement of LpxC activity (K_D^Fe) measured at 200 nm substrate, 30 °C, pH 7, as described under “Materials and Methods.” A binding isotherm is fit to these data. B, LpxC metal ion dissociation occurs readily in vitro. 10 μm ZnLpxC (closed circles) or FeLpxC (open circles) in 20 mm BisTris, pH 7.5, was incubated with varying concentrations of competing metal ion, and then bound metal ions were determined by ultrafiltration followed by ICP-MS analysis, as described under “Materials and Methods.” Dashed lines shown are simulations calculated from Equations 4 and 5 using the metal dissociation constants determined in A. C, 200 nm ZnLpxC (open circles), ZnLpxC + 100 μm palmitate (triangles), or FeLpxC (closed circles) was incubated in 1 mm EDTA, 20 mm BisTris, pH 7.5, 30 °C. At various times the deacetylase activity was measured by dilution (100-fold) into an assay containing 200 nm substrate, 1 mg/ml BSA, 20 mm BisTris, pH 7.5, 30 °C, as described under “Materials and Methods.” A single exponential decay is fit to the data. D, metal bound to LpxC can switch between Fe(II) and Zn(II) depending on the free metal ion concentrations. The stoichiometry of Fe(II) bound to LpxC (E·Fe/E_tot) was calculated as a function of [Zn]_free at 0.2, 0.4, 1, 3, or 6 μm [Fe]_free ([Fe]_free increases from the bottom to the top curve) using Equations 4 and 5 with the metal dissociation constants shown in Table 2.