Journal Article

Crystallographic snapshots of sulfur insertion by lipoyl synthase

Goldman, Peter J., McLaughlin, Martin I., Lee, Kyung-Hoon, Drennan, Catherine L., Lanz, Nicholas D., Booker, Squire J.

2016

Overview

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.

• Publisher: National Academy of Sciences
• Subject: Amino Acid Motifs / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / BASIC BIOLOGICAL SCIENCES / Biochemistry / Biological Sciences / Catalysis / Catalytic Domain / Chemistry / Cloning, Molecular / Crystallography / Crystallography, X-Ray / Escherichia coli - genetics / Escherichia coli - metabolism / Gene Expression / Genetic Vectors - chemistry / Genetic Vectors - metabolism / INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY / Iron - chemistry / Iron - metabolism / Iron-sulfur proteins / Iron-Sulfur Proteins - chemistry / Iron-Sulfur Proteins - genetics / Iron-Sulfur Proteins - metabolism / iron–sulfur cluster / Kinetics / lipoic acid / Models, Molecular / Mycobacterium tuberculosis - chemistry / Mycobacterium tuberculosis - enzymology / Peptides - chemistry / Peptides - metabolism / Physical Sciences / Protein Binding / Protein Conformation, alpha-Helical / Protein Conformation, beta-Strand / Protein Interaction Domains and Motifs / radical SAM enzyme / Recombinant Proteins - chemistry / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / S-Adenosylmethionine - chemistry / S-Adenosylmethionine - metabolism / Substrate Specificity / Substrates / Sulfur / Sulfur - chemistry / Sulfur - metabolism