Activation of Escherichia coli UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase by Fe2+ yields a more efficient enzyme with altered ligand affinity

Abstract

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.

Figure 1

(A) LpxC-catalyzed reaction. (B) Active site of LpxC from A. aeolicus containing two zinc ions: Zn_A (catalytic) and Zn_B (inhibitory). Figure was made from PDB 1P42. (C) Structure of L-161,240. (D) Structure of BODIPY-fatty acid.

Figure 2

Activation of apo-LpxC by Zn²⁺ or Fe²⁺. Deacetylase activity as a function of either Zn²⁺ (closed circles) or Fe²⁺ (open circles) metal ion stoichiometry was measured for WT LpxC (A) and C63A LpxC (B). C63A LpxC activity was assayed with up to 50-fold excess Fe²⁺ (not shown). The deacetylase activity for the substrate myr-UDPGlcNAc (0.2 μM) was measured at 30 °C after incubation with varying equivalents of M²⁺, as described under “Materials and Methods”.

Figure 3

Steady-state turnover catalyzed by EcC63A substituted with Zn²⁺ (filled circle) or Fe²⁺ (open circle). The initial rates for deacetylation of myr-UDPGlcNAc (0.05 – 4 μM) were measured at 30 °C in 20 mM bis-tris propane, 10 mM TCEP pH 7.5, as described under “Materials and Methods” using apo-enzyme reconstituted with stoichiometric metal ion. The parameters k_cat, K_M and k_cat/K_M (Table 1) were obtained by fitting the Michaelis-Menten equation to these data.

Figure 4

pH-dependence of LpxC-catalyzed deacetylase activity for Zn²⁺-C63A (●) or Fe²⁺ - C63A (○) LpxC. The values of V/K were measured at 30 °C using subsaturating concentrations of myr-UDPGlcNAc (≤ 0.2 μM) and enzyme reconstituted with stoichiometric metal ion, as described under “Materials and Methods”. The pK_a values (see Table 1) were determined by fitting Eq. 1 to the data.

Figure 5

XAS of Fe²⁺-EcLpxC. (A) XANES region. (B) Expansion of XANES showing 1s->3d transition with calculated background (blue) and best fit (green). Gaussian fit to 1s->3d transition is shown offset vertically for clarity. (C) k³ weighted EXAFS data (blue) together with best fit using 5 oxygen ligands (red, dashed). (D) Fourier transform of EXAFS data: experimental (solid line) and fit (dashed line) to 5 N/O ligands.

Figure 6

Dependence of LpxC activity on reducing agents. (A) The activity of Fe²⁺- and Zn²⁺-EcLpxC was measured at 30 °C in buffer containing either 10 mM (black bars) or 0.5 mM (gray bars) TCEP as described in the “Materials and Methods” section. (B) Apo-EcLpxC was reconstituted with either Fe²⁺ (open circle) or Zn²⁺ (filled circle) and the resulting activity was measured at 30 °C (20 mM bis-tris propane, 10 mM TCEP, pH 7.5) as a function of time.

Figure 7

Native LpxC activity. The LpxC deacetylase activity of E. coli crude cell lysates was measured at 30 °C as described in “Materials and Methods”. (A) LpxC activity of E. coli cell lysate measured in 20 mM bis-tris propane pH 7.5 containing 10 mM (black bars) or 0.1 mM TCEP (gray bars). (B) E. coli cell lysate activity assayed in 10 mM TCEP immediately following lysis (black bars) or after incubation on ice for 3 hours under aerobic (benchtop) conditions post-lysis (gray bars). (C) The LpxC inhibitor L-161,240 inhibits 90% of the deacetylase activity in the E. coli cell lysate (20 mM bis-tris propane, 10 mM TCEP pH 7.5). The concentration of L-161,240 was 0 μM (black bars) or (1 μM) (gray bars).

Figure 8

Proposed mechanism for EcLpxC.