title: Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid.
abstract: Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Cicchillo
authors.forename: David F
authors.initials: DF
authors.lastname: Iwig
authors.forename: A Daniel
authors.initials: AD
authors.lastname: Jones
authors.forename: Natasha M
authors.initials: NM
authors.lastname: Nesbitt
authors.forename: Camelia
authors.initials: C
authors.lastname: Baleanu-Gogonea
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Souder
authors.forename: Loretta
authors.initials: L
authors.lastname: Tu
authors.forename: Squire J
authors.initials: SJ
authors.lastname: Booker
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D003839:Deoxyadenosines / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006859:Hydrogen; D013058:Mass Spectrometry / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D008063:Thioctic Acid / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003839:Deoxyadenosines; C079999:LipA protein, Bacteria; D008063:Thioctic Acid; D012436:S-Adenosylmethionine; D006859:Hydrogen