The allosteric role of the AAA+ domain of ChlD protein from the magnesium chelatase of synechocystis species PCC 6803

Abstract

Magnesium chelatase is an AAA(+) ATPase that catalyzes the first step in chlorophyll biosynthesis, the energetically unfavorable insertion of a magnesium ion into a porphyrin ring. This enzyme contains two AAA(+) domains, one active in the ChlI protein and one inactive in the ChlD protein. Using a series of mutants in the AAA(+) domain of ChlD, we show that this site is essential for magnesium chelation and allosterically regulates Mg(2+) and MgATP(2-) binding.

Keywords: ATPases; Biosynthesis; Enzyme Catalysis; Mutagenesis Site-specific; Porphyrin.

FIGURE 1.

CD spectra (mean residue ellipticity) of His₆-ChlD variants (0. 1 mg ml⁻¹) at 25 °C in 5 mm sodium phosphate buffer, 1 mm β-mercaptoethanol, pH 7.4. Traces show wild type (○), K49A (□), E152Q (▵), R208A (♢), and R289A (●) His₆-ChlD.

FIGURE 2.

ATP-dependent complex formation between wild type ChlI (2 μm) and mutant His₆-ChlD (2 μm). All samples contained ChlI and in addition, lane 1, wild type His₆-ChlD and no ATP; lane 2, wild type His₆-ChlD with ATP; lane 3, His₆-ChlD (K49A) and no ATP; lane 4, His₆-ChlD (K49A) with ATP; lane 5, His₆-ChlD (E152Q) and no ATP; lane 6, His₆-ChlD (E152Q) with ATP; lane 7, His₆-ChlD (R208A) and no ATP; lane 8, His₆-ChlD (R208A) with ATP; lane 9, His₆-ChlD (R289A) and no ATP; lane 10, His₆-ChlD (R289A) with ATP.

FIGURE 3.

Chelatase assembly titrations between wild type ChlI and wild type (○), E152Q (▵), R208A (♢), and R289A (●) His₆-ChlD. Assays contained 0.1 μm ChlD, 0.4 μm ChlH in 50 mm MOPS/KOH, 0.3 m glycerol, 1 mm DTT, 10 mm free Mg²⁺, I = 0.1 with KCl, 8 μm D_IX, pH 7.7, 34 °C. The curves can be described by Equation 3 with the characterizing parameters: (wild type ChlD) K_app 0.17 ± 0.9 nm n_d 0.23 ± 0.01 μm and v_lim 0.22 ± 0.01 μm min⁻¹; (E152Q) K_app 0.35 ± 7.3 nm n_d 0.24 ± 0.01 μm and v_lim 0.04 ± 0.01 μm min⁻¹; (R208A) K_app 14 ± 11 nm n_d 0.20 ± 0.01 μm and v_lim 0.14 ± 0.01 μm min⁻¹; (R289A) K_app 52 ± 155 nm n_d 0.19 ± 0.09 μm and v_lim 0.016 ± 0.009 μm min⁻¹.

FIGURE 4.

MgATP²⁻ dependence of the steady-state chelation rate with wild type (a), R208A (b), E152Q (c), R289A (d), and K49A (e) variants of ChlD. Assays contained 0.1 μm ChlD, 0.2 μm ChlI, 0.4 μm ChlH in 50 mm MOPS/KOH, 0.3 m glycerol, 1 mm DTT, 10 mm free Mg²⁺, I = 0.1 with KCl, 8 μm D_IX, pH 7.7, 34 °C. The lines are described by Equation 2 with characterizing parameters k_cat 0.405 ± 0.014 min⁻¹ K_0.5 0.43 ± 0.02 mm, n 2.0 ± 0.2 (a); k_cat 0.263 ± 0.005 min⁻¹ K_0.5 0.78 ± 0.02 mm, n 2.0 ± 0.10 (b); k_cat 0.108 ± 0.004 min⁻¹ K_0.5 0.72 ± 0.04 mm, n 1.8 ± 0.17 (c); and by the Michaelis-Menten equation k_cat 0.054 ± 0.005 min⁻¹ K_m 1.79 ± 0.32 mm (d). No activity was observed with the K49A variant.

FIGURE 5.

Free magnesium dependence of the steady-state chelation rate with wild type (a), R208A (b), E152Q (c), and R289A (d) variants of ChlD. Assays contained 0.1 μm ChlD, 0.2 μm ChlI, 0.4 μm ChlH in 50 mm MOPS/KOH, 0.3 m glycerol, 1 mm DTT, 5 mm ATP, I = 0.1 with KCl, 8 μm D_IX, pH 7.7, 34 °C. The lines are described by Equation 2 with characterizing parameters k_cat 0.49 ± 0.01 min⁻¹ K_0.5 4.93 ± 0.04 mm, n 6.0 ± 0.3 (a); k_cat 0.50 ± 0.06 min⁻¹ K_0.5 7.18 ± 0.74 mm, n 2.4 ± 0.2 (b); k_cat 0.17 ± 0.02 min⁻¹ K_0.5 6.97 ± 0.66 mm, n 2.7 ± 0.3 (c); and by a straight line equation k_cat/K_m 1.7 ± 1.0 min⁻¹ mm⁻¹ (d).

FIGURE 6.

Deuteroporphyrin dependence of the steady-state chelation rate with wild type (a), R208A (b), E152Q (c), and R289A (d) variants of ChlD. Assays contained 0.1 μm ChlD, 0.2 μm ChlI, 0.4 μm ChlH in 50 mm MOPS/KOH, 0.3 m glycerol, 1 mm DTT, 5 mm ATP, I = 0.1 with KCl, pH 7.7, 34 °C. Lines are described by the Michaelis-Menten equation with the characterizing parameters k_cat 1.13 ± 0.06 min⁻¹ K_m 5.74 ± 0.77 μm (a); k_cat 0.86 min⁻¹, K_m 6.31 ± 0.75 μm (b); k_cat 0.20 ± 0.01 min⁻¹ K_m 1.23 ± 0.13 μm (c); and k_cat 0.08 ± 0.01 min⁻¹ K_m 0.99 ± 0.12 μm (d).