Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis
Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis
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DOI：
10.1074/jbc.m111.291591
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发表时间：
2011-09-30
影响因子：
4.8
通讯作者：
Smith, Stuart
中科院分区：
文献类型：
--
作者：
Witkowski, Andrzej;Thweatt, Jennifer;Smith, Stuart
本研究的目的是确定一个来源的线粒体内丙二酰辅酶A，可用于从头脂肪酸合成哺乳动物线粒体。由于哺乳动物线粒体缺乏能够在线粒体内产生丙二酰辅酶A的乙酰辅酶A羧化酶，因此研究了丙二酸可以作为前体的可能性。虽然丙二酰辅酶A合成酶尚未确定以前在动物中，动物蛋白质序列数据库的询问确定的候选人，表现出序列相似性，以已知的原核形式。人类候选蛋白ACSF 3，它具有预测的N-末端线粒体靶向序列，被克隆，表达，并表征为65-kDa的酰基辅酶A合成酶，具有极高的特异性丙二酸和甲基丙二酸。原核丙二酰辅酶A合成酶与丙二酸结合的精氨酸残基在动物ACSF 3酶中位置保守，对活性至关重要。HEK 293 T细胞的亚细胞分级实验证实，人ACSF 3仅位于线粒体中，RNA干扰实验证实，这种酶负责这些细胞线粒体中的大多数（如果不是全部）丙二酰辅酶A合成酶活性。总之，与具有线粒体内乙酰辅酶A羧化酶的真菌不同，动物需要线粒体丙二酰辅酶A的替代来源;线粒体ACSF 3酶能够通过利用游离丙二酸作为底物来填补这一角色。
The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.