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. 2020 Nov 10;86(23):e01359-20.
doi: 10.1128/AEM.01359-20. Print 2020 Nov 10.

A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Jian-Qiang Jin #  1 , Shin-Ichi Hachisuka #  1 , Takaaki Sato  1 , Tsuyoshi Fujiwara  1 , Haruyuki Atomi  2
Affiliations

A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Jian-Qiang Jin et al. Appl Environ Microbiol. .

Abstract

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea.IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.

Keywords: Archaea; Thermococcus; biosynthesis; cofactor; hyperthermophile; lipoic acid; lipoyl synthase; metabolism.

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Figures

FIG 1
FIG 1
The classical and proposed lipoic acid biosynthesis pathways. In the proposed pathway, LipA is replaced by LipS1/S2 identified in this study. An enzyme corresponding to LipB or LipM is still unclear in a number of archaeal species. “H” refers to H-protein in the glycine cleavage system. Lipoic acid-activating enzyme and lipoyl transferase are utilized in mammalian cells instead of LplA. Abbreviations: LipA, classical lipoyl synthase; LipB and LipM, octanoyl transferase; LplA, lipoate-protein ligase; LipS1/S2, archaeal lipoyl synthase.
FIG 2
FIG 2
Growth properties of the host strain KU216 and the gene disruption strains. Growth of the host KU216 strain (circles) and the ΔTK2109 (squares), ΔTK2248 (triangles), and ΔTK2109 ΔTK2248 (diamonds) mutants was examined in a synthetic amino acid medium without serine and lipoic acid (mASW-AA-Ser[−]-S0-Ura-Lip[−]) (A), in a synthetic medium supplemented with lipoic acid (mASW-AA-Ser[−]-S0-Ura-Lip[+]) (B), and in a medium supplemented with octanoic acid (mASW-AA-Ser[−]-S0-Ura-Oct[+]) (D). (C) Growth of the host KU216 strain (circles) and ΔTK2109 ΔTK2248 mutant (diamonds) in a synthetic medium (mASW-AA-S0-Ura) with (open symbols) or without (closed symbols) biotin. Error bars indicate the standard deviations of three independent culture experiments. The vertical axis is represented in logarithmic scale.
FIG 3
FIG 3
Gene arrangements of TK2109 and TK2248 homologs in seven archaea. Some archaeal organisms which possess TK2109 homolog, TK2248 homolog, and biotin or lipoate-protein ligase are shown. Vertical stripe, horizontal stripe, gray, and black arrowed boxes represent TK2109 homolog, TK2248 homolog, biotin or lipoate-protein ligase, and H-protein genes, respectively. Genes overlapping any of the above four genes, along with some neighboring genes, are shown with white arrowed boxes.
FIG 4
FIG 4
Lipoyl synthase activity measurement by HPLC analysis. HPLC analyses were performed with standard octanoyl-peptide (A), standard lipoyl-peptide (B), reduced lipoyl-peptide (C), reaction product without proteins (D), reaction product with reconstituted TK2109 protein (E), reaction product with reconstituted TK2248 protein (F), and reaction product with reconstituted TK2109 and TK2248 proteins (G). Abbreviations: OP, octanoyl-peptide; LP, lipoyl-peptide; RLP, reduced lipoyl-peptide; U1 to U4, unidentified compounds 1 to 4; AU, arbitrary units.
FIG 5
FIG 5
Identification of reaction products by LC-MS analysis. LC-MS analyses were carried out using reaction products with reconstituted TK2109 protein (A), reconstituted TK2248 protein (B), reconstituted TK2109 and TK2248 proteins (C), and nonreconstituted TK2109 and TK2248 proteins (D). Blue, red, green, and black lines represent the compounds whose m/z values corresponded to exact masses of octanoyl-peptide, lipoyl-peptide, reduced lipoyl-peptide, and intermediate thiol-octanoyl-peptide, respectively. Pep and RA, peptide and relative abundance, respectively.
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