Lipoyl Synthase Inserts Sulfur Atoms into an Octanoyl Substrate in a Stepwise Manner

Abstract

Lipoyl synthase (LipA) is required for the final step in the biosynthesis of lipoyl groups, the insertion of sulfur atoms at C6 and C8 of octanoyl groups (Scheme 1).[1] The octanoyl groups are found attached through an amide linkage to a lysine residue in a sequence motif that is conserved within a small family of protein domains [2] that include the H-protein of the glycine cleavage system [3] and the E2 subunit of oxoacid dehydrogenases. [4] After attachment of the octanoyl groups, [5][6][7] the substrates undergo sulfur insertion by LipA, [1] a member of the "radical S-adenosyl l-methionine" family.[8] Biochemical studies have shown that it contains two essential [4Fe-4S] clusters [9] and that one of these clusters is used to generate 5'-deoxyadenosyl radicals (AdoC) through the reductive cleavage of S-adenosyl l-methionine (AdoMet). The lipoyl forming reaction requires two hydrogen atoms to be removed from the octanoyl group, one from C6 and the other from C8, so that the overall reaction utilizes two equivalents of AdoMet to form each lipoyl group. [10] LipA has been shown to donate both of the inserted sulfur atoms [11] that were proposed to originate from a [4Fe-4S] cluster.[12] Recently, we reported that a short octanoyl peptide, which corresponds in sequence to the lipoylation site of the E2 subunit, could function as a substrate for LipA.[13] Peptide substrates have now been used to investigate the order of sulfur-insertion steps and hence clarify the structure of a key intermediate on the reaction pathway.The formation of lipoyl groups was monitored in a reaction by using Sulfolobus solfataricus P2 LipA with an octanoyl substrate tripeptide (0.5 mole equiv; 3, ), AdoMet, and dithionite as a reductant. LipA is not catalytic during assays in vitro, producing substoichiometric quantities of lipoyl products with either octanoyl-protein [10] or octanoylpeptide substrates.[13] To investigate the formation of any intermediate species, the reaction was stopped by acidification before reaching completion (after 20 min), the precipitated protein was pelleted by centrifugation, and the supernatant analyzed by LCMS. Four peptide species were eluted over the time range from 22 to 25 min (Table 1). The peptide at 24.4 min corresponds to the unreacted octanoyl substrate 3, whereas new species at 22.4 and 23.4 min correspond to the expected protonated product masses of lipoyl 5 and dihydrolipoyl products 4, respectively. The final species at 23.2 min coelutes with the dihydrolipoyl product and corresponds to a monothiolated species (either 6 or 7). Analysis of later time points (up to 2 h) showed that the amount of this monothiolated species decreased with time and that there was a Scheme 1. Insertion of sulfur atoms into octanoyl substrates to form lipoyl groups.

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…Mutagenesis has shown this serine ligand is essential for lipoyl product formation. In the context of biochemical and spectroscopic results [29,30], our results support a radical mediated mechanism of sulfur insertion at C6 and then C8 of the octanoyl chain, a process that leads to degradation of the sulfur donating auxiliary cluster. This aspect of the LipA mechanism resembles that proposed for biotin synthase [19,21], but differs significantly from the mechanism proposed for the methylthiotransferases RimO and MiaB [27].…”

“…A proposed mechanism for lipoyl synthase, including the highly unusual suggestion that the auxiliary cluster acts as a sacrificial sulfur donor, has been developed in the light of biochemical and spectroscopic studies [29,30,52,54]. Using a N Ɛ -octanoyl lysinyl peptide as a substrate analogue, a monothiolated species was isolated from activity assays and NMR analysis demonstrated that the sulfur insertion had occurred exclusively at C-6 (and not at C-8) [29].…”

“…Using a N Ɛ -octanoyl lysinyl peptide as a substrate analogue, a monothiolated species was isolated from activity assays and NMR analysis demonstrated that the sulfur insertion had occurred exclusively at C-6 (and not at C-8) [29]. This result is most simply explained in terms of sequential sulfur insertion reactions, firstly at C6 to form a 6-thiooctanoyl intermediate followed by a second sulfur insertion at C8 to complete the lipoyl cofactor.…”

“…N Ɛ -octanoyl lysine containing peptides have been used as model substrates for SsLipA to yield lipoyl peptides as products [28]. In addition, quenching of an activity assay with acid at an early time point led to the release of a 6-thiooctanoyl peptide, proposed as a monothiolated intermediate on the reaction pathway to the lipoyl product [29]. Recently, evidence for the catalytic and kinetically competent enzyme-substrate cross-linked intermediate of lipoyl synthase has been reported [30].…”

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

“…An octanoylated tetrapeptide suffices in the Sulfolobus solfataricus LipA assay (110). This system showed that LipA catalyzes lipoate biosynthesis in a stepwise manner, with sulfur first being inserted at C-6 of the octanoyl chain (109). Moreover, this intermediate remained tightly bound to LipA, consistent with the finding that both sulfur atoms are derived from the same LipA polypeptide.…”