Crystallographic snapshots of sulfur insertion by lipoyl synthase.
Publication Type:
Journal ArticleSource:
Proc Natl Acad Sci U S A, Volume 113, Issue 34, p.9446-50 (2016)Keywords:
Amino Acid Motifs, Bacterial Proteins, Catalytic Domain, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli, Gene Expression, Genetic Vectors, Iron, Iron-Sulfur Proteins, Kinetics, Models, Molecular, Mycobacterium tuberculosis, Peptides, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins, S-Adenosylmethionine, Substrate Specificity, SulfurAbstract:
<p>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.</p>