Evidence for a Catalytically and Kinetically Competent Enzyme–Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase

Abstract

Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe–4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5′-deoxyadenosyl 5′-radical (5′-dA•). In the LS reaction, two equivalents of 5′-dA• are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe–4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N6-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe–4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe–4S]0 clusters.

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

“…To complete the lipoyl cofactor, a second sulfur atom is required. In accord with biochemical results [30] and the active site steric constraints, loss of an iron atom from the auxiliary cluster is required to allow access to the second equivalent of sulfide to complete the lipoyl cofactor. It is likely that the serine ligand facilitates this loss of iron.…”

“…The auxiliary cluster has tentatively been proposed to act as the sulfur donor in the reaction [30,52]. Extending between the adenosyl moiety and auxiliary cluster is a 14 Å deep channel that is well-proportioned to accommodate the substrate N Ɛ -octanoyl lysine residue in an extended conformation.…”

“…The distance between octanoyl C6, the site of the substrate radical, and the potential sulfur donor, the proximal sulfur atom of the auxiliary cluster, is 6.4 Å. One of the other three sulfur atoms from the auxiliary cluster is proposed to be incorporated as the second atom of the lipoyl product [30,52], but the distance between these sulfides and the octanoyl C8 are 8.2, 8.4, 10.0 Å. For the C8 centered radical to react with one of these sulfide ions, the observed loss of an iron atom from the auxiliary cluster [30] may provide access, together with a conformational change in the octanoyl substrate.…”

“…One of the other three sulfur atoms from the auxiliary cluster is proposed to be incorporated as the second atom of the lipoyl product [30,52], but the distance between these sulfides and the octanoyl C8 are 8.2, 8.4, 10.0 Å. For the C8 centered radical to react with one of these sulfide ions, the observed loss of an iron atom from the auxiliary cluster [30] may provide access, together with a conformational change in the octanoyl substrate. On the opposite side of the auxiliary cluster from the octanoyl substrate binding site is the C-channel that stretches from Ser283 to the surface, a distance of over 11 Å, lying between the C-terminal loop (residues 283-288) and the β-sheet of the TIM barrel ( Figure 4D).…”

“…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]. The cross-link is proposed to result from a bond between the octanoyl C6 and the presumed sulfur donor, the auxiliary cluster, which is sacrificed during turnover.…”