Crystallographic snapshots of sulfur insertion by lipoyl synthase

Abstract

Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chemistry; the remainder of the reaction mechanism, especially the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resolution crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-containing 8-mer peptide substrate and 1 eq AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissociates from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.iron-sulfur cluster | radical SAM enzyme | lipoic acid T he functionalization of aliphatic carbon centers is widely regarded as one of the most kinetically challenging reactions in nature, a striking example of which is found in the biosynthesis of the lipoyl cofactor, famous for its central role as the "swinging arm" of the pyruvate dehydrogenase enzyme complex. Lipoyl synthase (LipA) generates the lipoyl cofactor by insertion of two sulfur atoms at C6 and C8 of a protein-bound n-octanoyl chain, sites distal from the nearest functionality (1-4). LipA and the closely related biotin synthase (BioB) (Scheme 1) are founding members of the ever-expanding S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily that uses a [4Fe-4S] cluster to reductively cleave the C5′-S bond of AdoMet, generating methionine and a 5′-deoxyadenosyl radical (5′-dA•), a powerful oxidant (5, 6). LipA requires 2 eq AdoMet-one per sulfur insertion-and two sulfur atoms to produce 1 eq lipoyl product through radical-based chemistry (4). One of the most controversial aspects of the LipA and BioB reactions is the source of the sulfur. AdoMet radical enzymes that catalyze sulfur insertion always seem to have an additional iron-sulfur (Fe/S) cluster: a [2Fe-2S] cluster in BioB (7), a [4Fe-4S] cluster in LipA (8), and a [4Fe-4S] cluster in the methylthiotransferases RimO and MiaB (9, 10). These auxiliary clusters in LipA and BioB have been proposed to be cannibalized during turnover to supply the inserted sulfur atom(s) (8, 11-13), a proposal that has not enjoyed univ...

Structural studies of viperin, an antiviral radical SAM enzyme

“…S3) and are similar to the binding modes observed in high-resolution crystal structures of other radical SAM enzymes (28,(38)(39)(40)(41)(42)(43) (Fig. S4).…”

Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase

“…3A is a reaction with 20 μM LipA containing Fe–S clusters composed of sulfide at natural abundance (~95% 32 S) and 100 μM NfuA containing Fe–S clusters composed of 34 S-labeled (~99%) sulfide. An initial burst of lipoic acid containing two 32 S sulfurs is observed (closed circles), which is consistent with previous results that indicate that the auxiliary cluster of LipA is used as the source of inserted sulfur atoms (19, 20, 40). Surprisingly, however, the amount of lipoic acid containing two 32 S atoms is more than 50% greater than that expected (33 μM vs. 20 μM), suggesting that the enzyme can potentially direct all four sulfide ions from the cluster into the lipoyl product rather than release two of them into solution during each turnover.…”

“…All radical SAM (RS) proteins contain one [4Fe–4S] cluster that supplies the electron during the reductive cleavage of SAM (22, 23). However, lipoyl synthases contain a second [4Fe–4S] cluster that has been hypothesized to be the source of the attached sulfur atoms (20, 24, 25). Consistent with this hypothesis, LipA typically catalyzes no more than one turnover in in vitro reactions due to the obligate destruction of the iron–sulfur (Fe–S) cluster (19, 21, 26).…”

“…The current working hypothesis for turnover by LipA is shown in Fig. 1 (19, 20, 27). SAM binds in contact with the RS [4Fe–4S] cluster— termed [4Fe–4S] RS —and undergoes reductive cleavage to generate a 5′-dA•, which abstracts an H• from C6 of the octanoyllysyl group of an LCP.…”

“…SAM binds in contact with the RS [4Fe–4S] cluster— termed [4Fe–4S] RS —and undergoes reductive cleavage to generate a 5′-dA•, which abstracts an H• from C6 of the octanoyllysyl group of an LCP. The resulting C6 radical attacks a bridging μ -sulfido ion of the auxiliary cluster with concomitant reduction of one of the Fe 3+ ions of the cluster to Fe 2+ and loss of an Fe 2+ ion to afford a [3Fe–3S–1(6 S )- thio-octanoyl-LCP] cluster (19, 20). A second 5′-dA•, generated from the reductive cleavage of a second SAM molecule, abstracts a C8 H•, with subsequent attack of the resulting C8 radical onto a second bridging μ -sulfido ion of the auxiliary cluster.…”