Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Abstract

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C₈-acyl carrier protein (C₈-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4'-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C₈-CoA. Furthermore, our ¹H saturation transfer difference and ³¹P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved ⁷¹RGG⁷³ loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

Keywords: ACP; C(8)-CoA; CoA; NMR; acyl carrier protein; glycine cleavage system H protein; lipoate protein ligase B; lipoic acid synthesis.

Figure 1

Backbone chemical shift perturbations of E. coli ACP upon LipB interaction.A, ribbon representation of M. tuberculosis LipB (PDB 1W66) covalently bound to a decanoyl-chain. The positively charged residues in the active site cavity are shown as sticks. M. tuberculosis residue numbering is shown in blue, and the corresponding E. coli numbering in black. B, a surface representation of M. tuberculosis LipB (colored based on coulombic charge), displaying the opening of the active site cavity. Changes in the amide chemical shift of (C) C₈-ACP, (E) holo-ACP, and (G) apo-ACP, upon titration with unlabeled LipBK135A/C169A. ACP backbone has been represented as a worm (PDB 5VCB, chain D) for (D) C₈-ACP, (F) holo-ACP, and (H) apo-ACP displaying chemical shift changes upon LipBK135A/C169A interaction. The magnitude of chemical shift change at each amide is directly proportional to the thickness of the worm. PDB, Protein Data Bank.

Figure 2

LipB can use noncognate ACP as well as C₈-CoA as octanoate donor.A, a 12% SDS-PAGE gel displaying lane 1: Apo-GcvH, lanes 2 to 4: C₈-ACP, C₈-LmACP, and C₈-PfACP loaded for reference, lane 5 molecular weight marker, lane 6 to 8: LipB assay using C₈-ACP (E. coli), C₈-LmACP (L. major), and C₈-PfACP (P. falciparum), respectively, as substrates. A1, a standard curve prepared based on the migration of standards in the SDS-PAGE gel of (A). The migration of E. coli ACP corresponds to a molecular weight of 18 kDa, LmACP ∼14 kDa, and PfACP ∼12 kDa. C18-reversed phase chromatogram for the GcvH conversion after the LipB assay performed using (B) C₈-EcACP, (C) C₈-LmACP, and (D) C₈-PfACP, as octanoate donors. The chromatogram before the assay is shown in black and after the assay in blue. %GcvH conversion is shown at the bottom of each chromatogram. E, a 12% SDS-PAGE gel displaying the conversion of GcvH, using different acyl-CoA as octanoate donors. Lane 2: apo-GcvH, Lane 3: assay in the absence of LipB, Lane 4 to 7: assay in presence of C₄-CoA, C₈-CoA, C₁₀-CoA, and C₁₂-CoA, respectively. F, a 12% SDS-PAGE gel, displaying the conversion of E. coli GcvH in presence of Mus musculus LipT2, using C₈-CoA as octanoyl donor. Lane 1: molecular weight marker, lane 2: apo-GcvH, lane 3: assay in the presence of mice LipT2. % conversion determined using HPLC is shown at the bottom of the lane.

Figure 3

LipB prefers C₈-CoA over C₈-ACP as a donor substrate.A, kinetic studies using -•- LipB, -▲- LipBR71A, -▪- LipBG72A/G73A, and -□-LipBK135A as octanoyl-transferases, C₈-CoA as a donor substrate, and GcvH as an acceptor substrate. Reported values are the average of two independent measurements done on a Symmetry C18 reversed phase HPLC column. The figure was drawn using GraphPad prism version 7.0. B, a 12% native-PAGE gel displaying lane 1 to 4: apo-ACP, C₈-ACP, holo-ACP, and apo-GcvH, respectively, used as controls. Lane 5 was loaded with LipB assay performed in presence of equimolar concentration of C₈-ACP and C₈-CoA used as donor substrates. Lane 6: assay done using C₈-ACP alone as a donor substrate. C, the same samples were loaded on a 12% SDS-PAGE gel. Lane 1: Marker, lane 2 to 5: apo-ACP, C₈-ACP, holo-ACP, and GcvH samples loaded as a reference. Lane 6: assay carried out using equimolar concentration of C₈-CoA and C₈-ACP, while lane 7 was loaded with the assay carried out in presence of C₈-ACP alone. In both the assays, GcvH was used as an acceptor substrate for the bisubstrate reaction, and its conversion to C₈-GcvH was followed by forward migration. C18-reversed phase chromatogram for (D) Apo-GcvH used as a reference, LipB assay in presence of (E) equimolar concentration of C₈-ACP: C₈-CoA, and (F) C₈-ACP alone. Samples in Figure (D–F) were the same samples that were loaded in lanes 4 to 6 of the Native-PAGE and lanes 5 to 7 of SDS-PAGE gels in figures (B) and (C).

Figure 4

LipB interacts with adenosine 3′5′-diphosphate and phosphopantetheine arm of CoA.A, the chemical structure of CoA is shown, displaying its two components phosphopantetheine (labeled brown) and ADP (labeled black). B1, ¹H NMR spectra of CoA in D₂O, (B2) ¹H spectra of CoA in Tris–HCl, (B3) ¹H spectra of CoA in Tris–HCl, in presence of 1 mM WT LipB. Saturation transfer difference (STD) spectra for (B4) CoA alone in buffer, (B5) CoA: wild LipB, (B6) CoA:LipBK135A/C169A, (B7) CoA:LipBG72A/G73A, and (B8) CoA:LipBR71A. C, ³¹P NMR spectra of (C1) WT LipB in Tris–HCl, (C2) CoA in Tris–HCl buffer, (C3) CoA: LipBK135A/C169A, (C4) CoA: WT LipB, (C5) CoA:LipBG72A/G73A, (C6) CoA:LipBC169A. The molar ratio of CoA:LipB was maintained at 10:1 in all STD experiments and 8:1 in the ³¹P NMR experiments.

Figure 5

RGG loop is necessary for full LipB activity.A, a 12% SDS-PAGE gel displaying molecular weight marker in lane 1, lane 2: apo-GcvH alone, lanes 3 to 7: assay carried out in presence of WT LipB, LipBG72A/G73A, LipBR71A, LipBK135A, and LipBK135A/C169A, respectively, using C₈-CoA as an octanoate donor. Percent conversion of GcvH after the assay is shown at the bottom of each lane. B, a 12% SDS-PAGE gel, displaying lane 1: marker, lane 2: apo-GcvH, lane 3 to 5: assay carried out with WT LipB, LipBG72A/G73A, and LipBR71A, using C₈-ACP as a donor substrate.

Figure 6

Molecular dynamics simulations of WT LipB and the mutants. Superposition of the 21 structures obtained at the end of GROMACS 20 ns simulations for (A) WT LipB, (B) LipBG77A/G78A, and (C) LipBR76A. D, the surface of wild type LipB is shown, displaying the active site cavity. E, root mean square fluctuation (CαRMSF) as a function of residue number, and (F) root-mean-square deviation (CαRMSD) for WT LipB (represented by black lines), LipBG77A/G78A (red line) and LipBR76A (green line) during the simulations.