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Review
. 2016 Apr 13;80(2):429-50.
doi: 10.1128/MMBR.00073-15. Print 2016 Jun.

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

John E Cronan  1
Affiliations
Review

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

John E Cronan. Microbiol Mol Biol Rev. .

Abstract

Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism.

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Figures

FIG 1
FIG 1
Lipoic acid and related molecules. Lipoic acid is a derivative of octanoic acid in which sulfur atoms are inserted at carbon atoms 6 and 8. The two thiols form the disulfide, which is called lipoic acid (dihydrolipoic acid is the reduced form). The IUPAC names of (R)-lipoic acid and dihydrolipoic acid are (R)-5-(1,2-dithiolan-3-yl)pentanoic acid and 6,8-dimercaptooctanoic acid, respectively.
FIG 2
FIG 2
The lipoate ligase reaction. The reaction proceeds in two partial reactions: the formation of lipoyl-adenylate, followed by transfer of the lipoate moiety to the acceptor protein (an E2 or GcvH protein). The lipoyl-adenylate intermediate is tightly bound. The biochemically characterized ligases also use octanoate as a substrate. Attachment is to an LD of a 2-oxoacid dehydrogenase E2 protein or to a glycine cleavage system H protein.
FIG 3
FIG 3
The lipoate-requiring enzymes. (A) The general 2-oxoacid dehydrogenase reaction. ThDP, thiamine diphosphate. (B) The glycine cleavage system (GCV, called glycine decarboxylase in plants). The synthesis of serine from N5,N10-methylene-tetrahydrofolate and a second molecule of glycine by serine hydroxymethyl transferase (SHMT) is not part of the glycine cleavage system but plays an important role in some organisms and may be a key factor in the first role of lipoic acid in metabolism. The E2 subunit of the 2-oxoacid dehydrogenases and the L protein of the glycine cleavage system are the same protein. (C) Structure of the LD of Bacillus stearothermophilus PDH E2 subunit (PDB code 1LAB).
FIG 4
FIG 4
The LipB reaction. The CoA-derived ACP prosthetic group is shown with the octanoate moiety in blue. AcpP (called AcpA in a few bacteria) is the protein moiety of ACP (ACP is defined as the species carrying the prosthetic group). B. subtilis LipM performs the same reaction via the same mechanism that utilizes a thioester-linked acyl-enzyme intermediate (Fig. 5). This is an unusual pathway in that one protein-bound prosthetic group, 4′-phosphopantheine, allows the synthesis of another, lipoic acid.
FIG 5
FIG 5
Comparison of the lipoate assembly and lipoic acid ligation pathways in E. coli. The top scheme shows the transfer of octanoate from ACP to the active site thiol of LipB, followed by the attack on the ε-amino group of the LD lysine residue on the LipB thioester to give octanoyl-LD. LipA then converts this intermediate to lipoate by the consumption of two molecules of S-adenosyl-l-methionine (SAM) and two sulfur atoms derived from the LipA auxiliary [Fe-S] cluster. Two molecules each of methionine (Met) and deoxyadenosine (DOA) are also produced. The bottom scheme shows the lipoate ligase reaction, as described in the legend to Fig. 2. Note that octanoate is also an LplA substrate.
FIG 6
FIG 6
The PFAM 03099 lipoate metabolism enzymes are constructed on a common scaffold. (A) Superimposition of the crystal structures (Cα) of the Mycobacterium tuberculosis LipB octanoyltransferase (PDB code 1W66) (in magenta) with the Thermoplasma acidophilum LplA lipoate ligase (PDB code 2C8M) (in cyan). Note that the structures are circularly permuted. An interesting model to generate circularly permuted proteins has been put forth for a PFAM 03099 biotin ligase (61). (B) Superimposition of the crystal structures (ribbon diagram) of Bacillus halodurans LipL (in green, PDB code 2P5I) with M. tuberculosis LipB (in blue, PDB code 1W66). Note the strong conservation of helices and sheets in both superimpositions.
FIG 7
FIG 7
The mechanistically diverse PF03099 lipoyl metabolism enzymes. (A) The lipoate ligase reaction catalyzed by B. subtilis LplJ and the LplA proteins of E. coli and S. coelicolor. (B) The octanoyltransferase reaction catalyzed by E. coli LipB and B. subtilis LipM. (C) The amidotransferase reaction catalyzed by the LipL proteins of B. subtilis and L. monocytogenes.
FIG 8
FIG 8
Bypass of LipB function by LplA-catalyzed attachment of exogenous octanoate to lipoyl domains (LDs) in E. coli.
FIG 9
FIG 9
The Thermosynechococcus elongates LipA structure (106). The auxiliary cluster (Aux) is to the left, and the radical SAM (RS) cluster is to the right. The SAM was modeled in based on the binding of the SAM decomposition product, methylthioadenosine, and the complex of another radical SAM enzyme with SAM, whereas the octanoate moiety was docked into a channel between the SAM molecule and the auxiliary cluster (106). The docking is supported by the demonstration that C-6 of octanoate is sufficiently close to the auxiliary cluster for sulfide extraction to occur (112). (Reprinted from reference with permission.)
FIG 10
FIG 10
The LipL amidotransferase reaction. The active-site thiol of LipL attacks the octanoyl (or lipoyl)-GcvH amide linkage to form an acyl enzyme intermediate, which is then attacked by the ε-amino group of the LD lysine residue. Note that the reactions are reversible, raising the possibility that lipoate could be shuttled among proteins, depending on metabolic conditions.
FIG 11
FIG 11
GcvH is required for 2-oxoacid dehydrogenase lipoylation. A strain of B. subtilis lacking the gcvH gene (ΔgcvH) failed to accumulate proteins that react with anti-lipoyl protein antibody when grown without lipoic acid (−LA). In contrast, when cells grown in the presence of lipoate are assayed, the E2 subunits of 2-oxoacid dehydrogenases are modified (all have molecular masses of 43 to 47 kDa).
FIG 12
FIG 12
Comparison of the lipoate assembly pathways of E. coli and B. subtilis.
FIG 13
FIG 13
The Listeria monocytogenes lipoate-scavenging pathway. Lpa denotes a lipoamidase activity encoded by an unknown gene(s) that cleaves lipoate from host proteins or peptides (91). This bacterium encodes two lipoate ligases, but only LplA1 functions during invasion of the host in pathogenesis (164). This is probably due to its more efficient utilization of lipoate (91). LplA1 is largely specific for modification of the H protein of the glycine cleavage system. Following attachment to GcvH, the lipoate can be transferred to the 2-oxoacid dehydrogenases. The transfer reaction has been shown to be reversible (91).
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