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.

Keywords: iron–sulfur cluster; lipoic acid; radical SAM enzyme.

Scheme 1.

The reactions catalyzed by the AdoMet radical sulfur insertion enzymes (Upper) BioB and (Lower) LipA. The inserted sulfur atoms are marked in red.

Fig. 1.

Structural comparisons of substrate-free and intermediate-bound forms of LipA. (A) Structure of substrate-free LipA in ribbon representation with the (β/α)₆ AdoMet radical domain (green), N-terminal extension (red), and C-terminal extension (blue). (B) The auxiliary [4Fe-4S] cluster of LipA in the absence of substrate. R290 and S292 of the R(S/T)SΦ motif are shown as sticks. Simulated annealing omit density (2F_o−F_c) is contoured at 2.0σ in black mesh. (C) Structure of LipA after reaction with 1 eq AdoMet in the presence of octanoyl-H peptide substrate (yellow); 5′-dA and methionine are in dark blue. A loop and helix comprising residues 4–30 become ordered when substrate is bound. (D) The auxiliary cluster of LipA with octanoyl-H peptide substrate (yellow) coordinated. Simulated annealing omit density (2F_o−F_c) is contoured at 2.0σ in black mesh.

Fig. 2.

The active sites of LipA and BioB. (A) Substrate-free LipA with R290 blocking access to auxiliary cluster. (B) Intermediate-bound LipA structure. (C) A cross-section of the intermediate-bound LipA structure showing water-filled cavity created by residues in β1 (E71–T73), β2 (Y117–T119), and the C-terminal helix (S292–R297). Inset depicts simulated annealing omit density (2F_o−F_c) around the auxiliary cluster contoured at 2.0σ. (D) BioB active site (Protein Data Bank ID code 1R30) with a highly conserved arginine residue, R260, ligating the auxiliary [2Fe-2S] cluster. AdoMet ligates the AdoMet radical cluster, whereas dethiobiotin, the substrate, is between AdoMet and the auxiliary cluster.

Fig. S1.

Overall fold of BioB and LipA. (A) Crystal structures of (Left) BioB and (Right) LipA in ribbon representation colored by domain. BioB adopts a (β/α)₈ full triosephosphate isomerase (TIM) barrel fold, whereas LipA contains the (β/α)₆ partial barrel more common among AdoMet radical enzymes, known as the AdoMet radical core fold. (B) Topology diagrams of (Left) BioB and (Right) LipA. Fe/S clusters are represented in ball and stick form. Fe/S cluster ligands are shown as circles and colored as follows: cyan, arginine; red, serine; and yellow, cysteine.

Fig. S2.

Sequence alignment of annotated LipAs from M. tuberculosis, E. coli, Thermus thermophilus, Sulfolobus solfataricus, Chlamydia trachomatis, Agrobacterium tumefaciens, Saccharomyces cerevisiae, and Homo sapiens truncated at residue 308 (M. tuberculosis numbering). Important residues are highlighted as follows: blue, R(T/S)SФ motif; dark gray, residues contacting 5′-dA and methionine; dark green, AdoMet radical CX₃CXФC motif; dark red, CX₄CX₅C motif; light gray, canonical AdoMet binding motifs; light green, AdoMet radical cluster ligands; light red, auxiliary cluster ligands; and yellow, residues contacting the octanoyllysine residue of substrate. Secondary structure is indicated above the sequence, with N standing for N-terminal extension and C standing for C-terminal extension. A corresponding topology diagram is shown in Fig. S1.

Scheme 2.

Proposed catalytic mechanism for LipA. Inserted sulfur atoms are shown in red; species represented by the crystal structures described in the text are shown in blue. The timing of dissociation of the conserved serine ligand, S292, is unknown, but it is shown concomitant with substrate binding. Modified from ref. .

Fig. 3.

Polar interactions between LipA and the octanoyl-H peptide substrate (yellow). Six chains are present in the crystal structure’s asymmetric unit with various degrees of order; the interactions shown are only those present in four or more chains and mediated by zero or one water molecule. The N-terminal extension is in red, the AdoMet radical core is in green, and the C-terminal extension is in cyan.

Fig. S3.

Conformational change in the N-terminal extension of LipA on substrate binding and formation of the covalent intermediate. An α-carbon difference distance matrix created using DDMP (Center for Structural Biology, Yale) shows changes in interresidue distances between the substrate-free and intermediate-bound structures. Pairs of residues that are closer to each other in the LipA intermediate structure are blue dots, whereas those that are closer in the substrate-free structure are red. The N-terminal extension (residues 4–70) is indicated in pink, the AdoMet radical core (residues 71–281) is in green, and the C-terminal extension (residues 282–311) is in cyan. Only the N-terminal extension moves substantially, with the C-terminal extension moving slightly as a result of crystal packing differences. The arrow indicates the direction of N-terminal extension movement on substrate binding and intermediate formation.

Fig. S4.

The active site of chain A of the LipA intermediate-bound structure. (A) Distances between C5′ of 5′-dA and its reaction partners and between C8 of substrate and its possible reaction partners. Other distances are listed in Table S2. (B) The observed stereochemistry of the LipA reaction—abstraction of the pro-R hydrogen atom and inversion of configuration at C6—is reflected in the intermediate-bound structure. Here, a riding hydrogen atom added to C6 is within van der Waals distance of C5′ of 5′-dA.