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.

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…This intermediate is a highly reactive primary radical capable of abstracting hydrogen atoms from unactivated carbon centers [12]. Consistent with this mode of action and the requirement to break two C-H bonds, the formation of lipoyl groups requires at least two equivalents of SAM [9]. Mutagenesis has identified the presence of two [4Fe-4S] clusters in LipA, each ligated by three cysteine residues [14].…”

“…Assembly of the octanoyl LCD is followed by insertion of sulfur atoms at C6 and C8 of the octanoyl chain [3]. The sulfur insertion reaction requires lipoyl synthase (LipA), a member of the radical SAM (RS) superfamily [3,9].…”

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

“…As mentioned above, lipoyl synthase is a member of the radical SAM enzyme superfamily, which utilizes a reduced iron-sulfur cluster and SAM to generate 5=-deoxyadenosyl radicals for further radical-based chemistry. In the lipoyl synthase reaction, the role of the 5=-deoxyadenosine radicals is to remove one hydrogen atom each from the C-6 and C-8 positions of the octanoyl moiety, thereby allowing for subsequent sulfur insertion (98,108). Consistent with this prediction, two molecules of SAM are required to synthesize one molecule of lipoyl cofactor (108).…”

The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

“…The reaction by the sQhpC C7S ⅐QhpD complex continued linearly over 60 min for production of both methionine and 5Ј-dA with a multiple-turnover rate constant (k cat ) of about 0.193 min Ϫ1 at 1 mM SAM, when measured immediately after reconstitution (Fig. 8B) (39). By measuring the initial rates of 5Ј-dA production at different SAM concentrations, the apparent K m value for SAM was determined to be 45.3 Ϯ 5.4 M. The observation that the rate of 5Ј-dA production with the wild-type sQhpC⅐QhpD complex was slightly higher than that with sQhpC C7S ⅐QhpD (Fig.…”