title: Characterization of Lipoyl Synthase from Mycobacterium tuberculosis.
abstract: The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe-4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that observed in the E. coli LipA reaction. In addition, we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of association is dependent on the presence of S-adenosyl-l-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine.
authors: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: Nicholas D
authors: ND
authors: Lanz
authors: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: Kyung-Hoon
authors: KH
authors: Lee
authors: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: Abigail K
authors: AK
authors: Horstmann
authors: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: Maria-Eirini
authors: ME
authors: Pandelia
authors: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: Robert M
authors: RM
authors: Cicchillo
authors: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: Carsten
authors: C
authors: Krebs
authors: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §The Howard Hughes Medical Institute, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: Squire J
authors: SJ
authors: Booker
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013466:Sulfurtransferases / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D013466:Sulfurtransferases; C116435:lipoic acid synthase