A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

Abstract

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl-GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester-linked acyl-enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys-Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.

Fig. 1

Panel A: Sequence coverage of LipL by LC-MS/MS analysis of LipL derived peptdides. The LipL peptide sequences detected are underlined. Panel B: Modifications of LipL detected after trypsin digestion followed by LC-MS/MS analysis. The theoretical peptides are shown with the modified cysteine residues in bold and underlined. The mass differences of the modification are given. The ion score is equal to −10log(P) where P is the probability the result is random chance

Fig. 2

Specificity of B. subtilis LipM, B. subtilis LplJ, and E. coli LipB for various lipoyl domains. In panels A and C the lipoyl domain tested as a substrate is indicated at the top of each lane and by an arrow. The [1-¹⁴C]octanoyl-ACP substrate is indicated by an arrow. Autoradiograms of SDS-PAGE gel separations of lipoyl domain substrate specificity assays are shown. Sodium [1-¹⁴C]octanoate was used as a substrate for LplJ. Panel A: LipM octanoyltransfer to lipoyl domains from [1-¹⁴C]octanoyl-ACP generated using AasS. Panel B: Ligation of [1-¹⁴C]octanoate by LplJ to lipoyl domains. Panel C: LipB octanoyltransfer to lipoyl domains from [1-¹⁴C]octanoyl-ACP generated using Vibrio harveyi acyl-ACP synthetase (AasS). The two E2 domains migrate similarly and are denoted by E2s.

Fig. 3

Panel A: Component controls for a coupled AasS/LipM/LipL assay starting with [1-¹⁴C]octanoate which becomes ligated to ACP by AasS. Following the reaction the proteins were resolved by SDS-PAGE and visualized by autoradiography. The identities of the [1-¹⁴C]octanoylated proteins are indicated. Panel B: Component controls for a coupled LplJ plus LipL assay as in Panel A. The starting substrates for LplJ–catalyzed ligation reactions were [1-¹⁴C]octanoate and ATP.

Fig. 4

Autoradiograms of SDS-PAGE gels of assays for LipL-catalyzed amidotransfer from purified [1-¹⁴C]octanoyl-GcvH to lipoyl domains. Panel A: Amidotransfer of the [1-¹⁴C]octanoyl moiety from purified octanoyl-GcvH to the unmodified lipoyl domain indicated. Each reaction contained purified wild type (WT) LipL. The two E2 domains migrate similarly and are denoted by E2s. Panel B: Amidotransfer from purified [1-¹⁴C]octanoyl-GcvH to the E2_PdhC. The purified wild type LipL or point mutant proteins indicated were used as enzyme sources. Panel C: Additional enzyme was added to allow detection of the octanoyl-LipL intermediate. Wild type (WT), C150A point mutant, C150S point mutant LipL were assayed in addition to a control (NE) lacking LipL. Panel D: Schematic of the LipL amidotransfer reaction is shown with the acyl-LipL intermediate.

Fig. 5

Alignment of members of the LipL clade. Positions having 50% or greater similarity are highlighted in grey. The catalytic cysteine residue (C150 in B. subtilis LipL), the other modified cysteine residue (C39 in B. subtilis LipL), and the conserved PF03099 lysine residue are highlighted in black. BACSU is Bacillus subtilis 168, BACHD is Bacillus halodurans, LISMO is Listeria monocytogenes, LACBA is Lactobacillus brevis and STRPC is Streptococcus pyogenes serotype M12 (strain MGAS9429). This alignment is part of the larger one used to create Fig. 8A.

Fig. 6

Panel A: Autoradiograph of an SDS-PAGE gel of octanoyltransfer assays using AasS generated [1-¹⁴C]octanoyl-ACP as a substrate. Extracts of the mutant strains indicated were used as a source of enzyme for transfer to GcvH and PdhC. All strains also carried a disruption in lplJ. When purified LipM and LipL were added to replace the missing protein(s) this is indicated with a plus sign.

Fig. 7

Growth and lipoylation phenotypes of a B. subtilis ΔgcvH strain. Growth of strains on minimal glucose media plates with the supplements indicated after 48 h at 37°C. LA denotes lipoic acid. Panel A: Growth of strains JH642 (wild type) and NM20 (ΔgcvH). Panel B: Growth of strains JH642 (wild type) and NM21 (ΔgcvH amyE:: Pspac-gcvH). Panel C: Lipoylation of GcvH in B. subtilis crude extracts. SDS-PAGE and immunoblotting with anti-LA antibody was done as described in Experimental Procedures. Cells were grown 22 h in minimal media with or without lipoate added as indicated. Crude extracts of B. subtilis wild type (WT) and ΔgcvH strains were used as indicated.

Fig. 8

Minimum evolution analysis of alignments with bootstrap percentage values shown for each branch. The trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The scale represents a 50% difference in compared residues per length. Branches representing Bacillus proteins are indicated in bold. Panel A: Analysis of selected catalytic domains from PF03099 the structurally related biotin ligases are used as the out-group. Panel B: Selected lipoyl domains involved in lipoic acid metabolism are shown.

Fig. 9

Structure of LipL and comparison with that of LipB. Panel A: The unpublished structure of B. halodurans LipL (PDB 2P5I). The modified cysteine residues, C39 and C150, detected by LC-MS/MS of the B. subtilis protein are shown in red. Panel B: Structural alignment of LipL from B. halodurans with M. tuberculosis LipB (PDB 1W66) (Ma et al., 2006). The active site adduct is shown in purple. Panel C: Close up view of the structural overlay with the LipB decanoyl adduct removed. The active site cysteine sulfur atoms are colored orange whereas carbon atoms are white. The distance between the two sulfur atoms is 2.4 Å.

Fig. 10

Current models for lipoic acid biosynthesis in E. coli and B. subtilis. Lipoic acid synthesis in E. coli is accomplished by two enzymes whereas B. subtilis requires three enzymes. B. subtilis also requires GcvH as an intermediate carrier whereas E. coli does not. We lack data to determine if LipA acts before or after LipL. We propose that B. subtilis, LipA uses octanoyl-LD (both octanoyl-GcvH and octanoyl-E2) as substrates, and therefore can act before or after LipL.