Presentation Information
[P01-029]Identification of lipoic acid biosynthesis system in hyperthermophilic archaea
○Takaaki SATO1,2, Jian-Qiang JIN1, Shin-ichi HACHISUKA3, Tsuyoshi FUJIWARA4, Haruyuki ATOMI1,2 (1. Graduate School of Engineering, Kyoto University (Japan), 2. Integrated Research Center for Carbon Negative Science, Kyoto University (Japan), 3. Graduate School of Engineering, Hokkaido University (Japan), 4. Faculty of Engineering, Kyoto University (Japan))
Keywords:
Archaea,Lipoic acid,Cofactor,Biosynthesis,Enzyme
[Purpose]
Lipoic acid is a C8 fatty acid containing two sulfur atoms at C-6 and C-8 carbon positions and a cofactor for C1 metabolism including glycine cleavage system (GCS)(a). It is found in all three domains, Bacteria, Eucarya, and Archaea, and is generally produced as a lipoyl group on proteins such as the H-protein in GCS. However, its biosynthesis system has been unclear in many hyperthermophilic archaea. Especially, (1) lipoyl synthase catalyzing sulfur insertion reaction was missing enzyme, (2) scaffold protein(s) to generate Fe-S cluster for lipoyl synthase reaction was not identified, (3) physiological function of lipoate-protein ligase, considered to be involved in lipoic acid salvage, was unclear. In this study, we aimed to identify lipoic acid biosynthesis system in the hyperthermophilic archaeon Thermococcus kodakarensis.
[Method & Results]
Genomic information suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase, are not involved in biotin biosynthesis. Based on the similarity in reaction chemistry between biotin synthase and lipoate synthase, proteins encoded by these genes were predicted to act as lipoyl synthase. We carried out genetic analysis of TK2109 and TK2248 and found that these genes contribute to lipoate biosynthesis. The purified recombinant proteins exhibited lipoyl synthase activity toward an octanoyl-octapeptide synthesized chemically. These in vivo and in vitro experiments indicated that TK2109 and TK2248 genes encode a structurally novel lipoyl synthase(b). The lipoyl synthases require iron-sulfur (Fe-S) cluster. We found that TK0731 and TK0730 proteins display middle and low similarity with previously-identified scaffold proteins, respectively. Functional analysis revealed that TK0731 and TK0730 recombinant proteins can form a scaffold protein complex for Fe-S cluster generation and transfer the cluster to TK2109 and TK2248 to catalyze lipoyl synthase reaction(c). TK1908 and TK1234 are annotated as the N- and C-terminal regions of lipoate-protein ligase (Lpl), respectively. Their recombinant proteins together displayed significant ligase activity toward octanoate in addition to lipoate when a peptide, a portion of H-protein, was used as an acceptor. It is noted that the gene disruption of TK1908 led to serine auxotrophy. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results implied that these proteins also function in de novo lipoate biosynthesis, most likely by ligating octanoate and H-protein(d). The results obtained in these studies figured out the previously-unidentified lipoic acid biosynthesis system.
[References]
(a) Cronan JE. Microbiol. Mol. Biol. Rev. 80: 429-50 (2016)
(b) Jin JQ, Hachisuka SI, Sato T, Fujiwara T, Atomi H. Appl. Environ. Microbiol. 86: e01359-20 (2020)
(c) Jin JQ, Sato T, Atomi H. Appl. Environ. Microbiol. 91: e0143825 (2025)
(d) Jin JQ, Sato T, Hachisuka SI, Atomi H. Appl. Environ. Microbiol. 88: e0064422 (2022)
Lipoic acid is a C8 fatty acid containing two sulfur atoms at C-6 and C-8 carbon positions and a cofactor for C1 metabolism including glycine cleavage system (GCS)(a). It is found in all three domains, Bacteria, Eucarya, and Archaea, and is generally produced as a lipoyl group on proteins such as the H-protein in GCS. However, its biosynthesis system has been unclear in many hyperthermophilic archaea. Especially, (1) lipoyl synthase catalyzing sulfur insertion reaction was missing enzyme, (2) scaffold protein(s) to generate Fe-S cluster for lipoyl synthase reaction was not identified, (3) physiological function of lipoate-protein ligase, considered to be involved in lipoic acid salvage, was unclear. In this study, we aimed to identify lipoic acid biosynthesis system in the hyperthermophilic archaeon Thermococcus kodakarensis.
[Method & Results]
Genomic information suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase, are not involved in biotin biosynthesis. Based on the similarity in reaction chemistry between biotin synthase and lipoate synthase, proteins encoded by these genes were predicted to act as lipoyl synthase. We carried out genetic analysis of TK2109 and TK2248 and found that these genes contribute to lipoate biosynthesis. The purified recombinant proteins exhibited lipoyl synthase activity toward an octanoyl-octapeptide synthesized chemically. These in vivo and in vitro experiments indicated that TK2109 and TK2248 genes encode a structurally novel lipoyl synthase(b). The lipoyl synthases require iron-sulfur (Fe-S) cluster. We found that TK0731 and TK0730 proteins display middle and low similarity with previously-identified scaffold proteins, respectively. Functional analysis revealed that TK0731 and TK0730 recombinant proteins can form a scaffold protein complex for Fe-S cluster generation and transfer the cluster to TK2109 and TK2248 to catalyze lipoyl synthase reaction(c). TK1908 and TK1234 are annotated as the N- and C-terminal regions of lipoate-protein ligase (Lpl), respectively. Their recombinant proteins together displayed significant ligase activity toward octanoate in addition to lipoate when a peptide, a portion of H-protein, was used as an acceptor. It is noted that the gene disruption of TK1908 led to serine auxotrophy. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results implied that these proteins also function in de novo lipoate biosynthesis, most likely by ligating octanoate and H-protein(d). The results obtained in these studies figured out the previously-unidentified lipoic acid biosynthesis system.
[References]
(a) Cronan JE. Microbiol. Mol. Biol. Rev. 80: 429-50 (2016)
(b) Jin JQ, Hachisuka SI, Sato T, Fujiwara T, Atomi H. Appl. Environ. Microbiol. 86: e01359-20 (2020)
(c) Jin JQ, Sato T, Atomi H. Appl. Environ. Microbiol. 91: e0143825 (2025)
(d) Jin JQ, Sato T, Hachisuka SI, Atomi H. Appl. Environ. Microbiol. 88: e0064422 (2022)
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