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

Abstract

Lipoic acid is an eight-carbon sulfur-containing biomolecule that functions primarily as a cofactor in several multienzyme complexes. It is biosynthesized as an attachment to a specific lysyl residue on one of the subunits of these multienzyme complexes. In Escherichia coli and many other organisms, this biosynthetic pathway involves two dedicated proteins: octanoyltransferase (LipB) and lipoyl synthase (LipA). LipB transfers an n-octanoyl chain from the octanoyl-acyl carrier protein to the target lysyl residue, and then, LipA attaches two sulfur atoms (one at C6 and one at C8) to give the final lipoyl cofactor. All classical lipoyl synthases (LSs) are radical S-adenosylmethionine (SAM) enzymes, which use an [Fe4S4] cluster to reductively cleave SAM to generate a 5′-deoxyadenosyl 5′-radical. Classical LSs also contain a second [Fe4S4] cluster that serves as the source of both appended sulfur atoms. Recently, a novel pathway for generating the lipoyl cofactor was reported. This pathway replaces the canonical LS with two proteins, LipS1 and LipS2, which act together to catalyze formation of the lipoyl cofactor. In this work, we further characterize LipS1 and LipS2 biochemically and spectroscopically. Although LipS1 and LipS2 were previously annotated as biotin synthases, we show that both proteins, unlike E. coli biotin synthase, contain two [Fe4S4] clusters. We identify the cluster ligands to both iron–sulfur clusters in both proteins and show that LipS2 acts only on an octanoyl-containing substrate, while LipS1 acts only on an 8-mercaptooctanoyl-containing substrate. Therefore, similarly to E. coli biotin synthase and in contrast to E. coli LipA, sulfur attachment takes place initially at the terminal carbon (C8) and then at the C6 methylene carbon.

Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe–5S Cluster for Sulfur Insertion

“…33 A recent publication demonstrated that two enzymes from Thermococcus kodakarensis (LipS1 and LipS2), previously annotated as biotin synthases, have 4Fe−4S auxiliary clusters and work together to catalyze the formation of lipoyl groups on an octanoyl-lysyl-containing peptide substrate. 42 However, no current crystal structure for these enzymes exists, and Mossbauer spectroscopy data from this study cannot distinguish whether a sulfur atom is ligated to the 4Fe−4S auxiliary cluster. We believe that these novel lipoyl synthases will utilize a 4Fe−5S cluster as the sulfur source, similar to Type II BioBs.…”

“…This is based on the facts that LipS1 and LipS2 have an absolutely conserved His residue that is required for catalysis and a nearby hydrogen bond donor (serine) that are both structurally adjacent to the predicted binding site of the auxiliary 4Fe−4S cluster. 42 Due to this misannotation and the similarity between LipS1 and LipS2 with Type II BioB, this opens the possibility of an alternative substrate for boBioB. Since lipoate is mainly used for metabolism in aerobic organisms, many anaerobic bacteria do not contain a lipoyl synthase gene, 43 and as expected, there is no gene encoding for lipoyl synthase that can be found within published Blautia obeum genomes (NC_021022.1, NZ_CYZA01000015.1).…”

“…We believe that these novel lipoyl synthases will utilize a 4Fe–5S cluster as the sulfur source, similar to Type II BioBs. This is based on the facts that LipS1 and LipS2 have an absolutely conserved His residue that is required for catalysis and a nearby hydrogen bond donor (serine) that are both structurally adjacent to the predicted binding site of the auxiliary 4Fe–4S cluster . Due to this misannotation and the similarity between LipS1 and LipS2 with Type II BioB, this opens the possibility of an alternative substrate for boBioB.…”

Twenty Years of Radical SAM! The Genesis of the Superfamily

“…The work by Booker and colleagues showed that like the reaction catalyzed by Escherichia coli BioB, sulfur insertion takes place first at the terminal carbon of the substrate, while in canonical lipoyl synthases sulfur insertion takes place first at the internal (C6) carbon (Figure ). Unlike E. coli BioB, however, both LipS1 and LipS2 contain auxiliary [Fe 4 S 4 ] clusters …”

“…Unlike E. coli BioB, however, both LipS1 and LipS2 contain auxiliary [Fe 4 S 4 ] clusters. 63 One of the areas of RS enzymology that highlights the extreme power and versatility of these enzymes is in the generation of complex metallocofactors, such as the H-cluster of the [FeFe]-hydrogenase and the molybdenum−iron (MoFe) cofactor of nitrogenase (Figure 8). The [FeFe] hydrogenase is one of three subclasses of hydrogenases, which catalyze the reversible reduction of protons to H 2 .…”