Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid
Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid
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DOI：
10.1021/bi049528x
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发表时间：
2004-06-01
期刊：
影响因子：
2.9
通讯作者：
Booker, SJ
中科院分区：
文献类型：
--
作者：
Cicchillo, RM;Iwig, DF;Booker, SJ
脂酰合酶（利帕）催化硫辛酰辅因子的形成，硫辛酰辅因子被几种多酶复合物用于各种α-酮酸的氧化脱羧，以及甘氨酸裂解成CO2和NH3，伴随着其α-碳转移到四氢叶酸，产生N-5，N-10-亚甲基四氢叶酸。在每种情况下，硫辛酰辅因子以酰胺键共价连接至位于复合物的指定硫辛酰承载亚基上的保守赖氨酸残基。遗传和生物化学研究表明，硫辛酰合酶是新建立的一类金属酶的成员，该金属酶使用S-腺苷-L-甲硫氨酸（S-Met）作为5 ′-脱氧腺苷自由基（5 ′-dA（.））的来源，它是每个反应中的专性中间体。这些酶含有铁-硫簇，其在α-Met裂解期间提供电子，除了初级自由基之外还形成L-甲硫氨酸。最近，已显示硫辛酰合酶的一种底物是丙酮酸脱氢酶复合物的携带硫辛酰的亚基（E-2）的辛酰化衍生物[Zhao，S.，米勒，J.R.，Jian，Y.，Marletta，M.一、和Cronan，J.E.，Jr.等人（2003）Chem.Biol.10，1293-1302]。在此，我们表明，辛酰化衍生物的辛酰轴承亚基的甘氨酸裂解系统（H-蛋白）也是利帕的底物，提供了进一步的证据，辅因子是合成其靶蛋白。此外，我们还证明了5 '-dA（.）直接作用于辛酰基底物，如氘从[辛酰基-d（15）] H-蛋白转移到5 '-脱氧腺苷所证明的。最后，我们的数据表明，2当量的LipA Met在形成1当量的[硫辛酰基] H-蛋白时被不可逆地切割，并且与其中需要两个利帕蛋白来合成一个硫辛酰基的模型一致。
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 CO2 and NH3, 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.