Zhyvoloup Alexander, Nemazanyy Ivan, Babich Aleksei, Panasyuk Ganna, Pobigailo Natalya, Vudmaska Mariya, Naidenov Valeriy, Kukharenko Oleksandr, Palchevskii Sergiy, Savinska Liliya, Ovcharenko Galina, Verdier Frederique, Valovka Taras, Fenton Tim, Rebholz Heike, Wang Mong-Lien, Shepherd Peter, Matsuka Genadiy, Filonenko Valeriy, Gout Ivan T
Department of Structure and Function of Nucleic Acid, The Institute of Molecular Biology and Genetics, 150 Zabolotnogo Street, Kyiv 143, Ukraine.
J Biol Chem. 2002 Jun 21;277(25):22107-10. doi: 10.1074/jbc.C200195200. Epub 2002 Apr 29.
Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.
辅酶A在活细胞中作为乙酰基和酰基的载体发挥作用，对众多生物合成、能量产生和降解代谢途径至关重要。辅酶A生物合成有五个酶促步骤。迄今为止，关于哺乳动物辅酶A生物合成途径中相关酶的分子克隆，仅报道了泛酸激酶。在本研究中，我们展示了辅酶A合成酶的cDNA克隆及功能特性。它有一个563个氨基酸的开放阅读框，编码一个约60 kDa的蛋白质。序列比对表明该蛋白质同时具有磷酸泛酰巯基乙胺腺苷酰转移酶和脱磷酸辅酶A激酶结构域。使用野生型重组蛋白的生化分析证实该基因产物确实含有这两种酶活性。通过定点诱变进一步证实了内在磷酸泛酰巯基乙胺腺苷酰转移酶活性的存在。因此，本研究描述了首个哺乳动物辅酶A合成酶的克隆与特性分析，并证实这是一种包含辅酶A生物合成最后两个组分的双功能酶。
