A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Abstract

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 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 C6 and C8 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. Herein, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that both TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are 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 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 in 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.

Characterization of LipS1 and LipS2 from Thermococcus kodakarensis: Proteins Annotated as Biotin Synthases, which Together Catalyze Formation of the Lipoyl Cofactor

“…Recently, Atomi and co-workers showed that both LipS1 (TK2109) and LipS2 (TK2248) from the hyperthermophilic archaeon Thermococcus kodakarensis are involved in lipoic acid biosynthesis . This organism does not contain a gene encoding a classical LS but contains genes that were annotated as encoding biotin synthases.…”

“…Recently, Atomi and co-workers showed that both LipS1 (TK2109) and LipS2 (TK2248) from the hyperthermophilic archaeon Thermococcus kodakarensis are involved in lipoic acid biosynthesis. 44 This organism does not contain a gene encoding a classical LS but contains genes that were annotated as encoding biotin synthases. They also showed, using a chemically synthesized octanoyllysine-containing octapeptide corresponding to a sequence of the H protein of the GCS, that the recombinant proteins, only when together, catalyze the formation of the lipoyl group, suggesting that one protein attaches sulfur at C8, while the second attaches sulfur at C6.…”

“…Many archaea that encode canonical LSs also encode the E2 subunit of the PDC, suggesting that the purpose of lipoic acid biosynthesis might be to generate the lipoyl group for occasions when the organism is undergoing aerobic metabolism. By contrast, relatively few archaea that encode LipS1 and LipS2 also encode the E2 subunit of the PDC, raising the question of the role of lipoic acid in these organisms …”

“…By contrast, relatively few archaea that encode LipS1 and LipS2 also encode the E2 subunit of the PDC, raising the question of the role of lipoic acid in these organisms. 44 ■ ASSOCIATED CONTENT * sı Supporting Information…”

“…32 Recently, it was reported that the genome of Thermococcus kodakarensis does not encode a canonical lipoyl synthase but instead uses two gene products in tandem (LipS1 and LipS2) to perform each of the sulfur attachment steps. 44 Interestingly, LipS1 and LipS2 were annotated as biotin synthases, but the isolated proteins, when together, were shown to generate a lipoyl group on an octanoyllysyl-containing peptide substrate surrogate. In T. kodakarensis, LipS1 and LipS2 are not clustered; they are >130 genes apart on the chromosome.…”