title: 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.
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; Department of Chemistry, The Pennsylvania State University, University Park, PA 16802;
authors: Martin I
authors: MI
authors: McLaughlin
authors: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802;
authors: Nicholas D
authors: ND
authors: Lanz
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors: Peter J
authors: PJ
authors: Goldman
authors: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802;
authors: Kyung-Hoon
authors: KH
authors: Lee
authors: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802; Howard Hughes Medical Institute, The Pennsylvania State University, University Park, PA 16802;
authors: Squire J
authors: SJ
authors: Booker
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139 cdrennan@mit.edu.
authors: Catherine L
authors: CL
authors: Drennan
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013455:Sulfur / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C079999:LipA protein, Bacteria; D010455:Peptides; D011994:Recombinant Proteins; D013455:Sulfur; D012436:S-Adenosylmethionine; D007501:Iron
keywords: iron–sulfur cluster; lipoic acid; radical SAM enzyme