Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello, Università degli Studi di Bari, Via Orabona 4, I-70126 Bari, Italy.
Mitochondrion. 2010 Apr;10(3):263-73. doi: 10.1016/j.mito.2009.12.149. Epub 2010 Jan 7.
FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent K(m) and V(max) values for FMN equal to 1.3+/-0.7 microM and 4.4+/-1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 microM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria.
黄素腺嘌呤二核苷酸合成酶或 ATP:FMN 腺苷酰转移酶（FADS 或 FMNAT，EC 2.7.7.2）是将核黄素转化为氧化还原辅酶 FAD 的代谢途径中的关键酶。我们在这里面临的是真核生物中 FADS 的亚细胞定位仍然存在争议的问题。首先，通过 Western blot 实验，我们证实了大鼠肝中存在不同的 FADS 同工型，它们在分子量和亚细胞定位上是不同的。SDS-PAGE 上的一条具有明显 60 kDa 分子量的交叉反应带定位于新鲜分离的纯化大鼠肝线粒体的内部隔室中。最近，我们在人类中鉴定了两种 FADS 同工型，它们在 N 端有 97 个氨基酸的额外序列不同，仅存在于同工型 1（hFADS1）中。hFADS1 的前 17 个残基代表一个可切割的线粒体靶向序列（通过 Target-P 预测）。在大肠杆菌中产生的重组 hFADS1 对 FMN 的表观 K(m)和 V(max)值分别为 1.3+/-0.7 microM 和 4.4+/-1.3 nmol x min(-1) x mg 蛋白(-1)，并且在高于 1.5 microM 的浓度时被 FMN 抑制。体外合成的 hFADS1，但不是 hFADS2，被导入大鼠肝线粒体并加工成较低分子量的蛋白质产物。在瞬时表达两种人类同工型的 BHK-21 和 Caco-2 细胞系上进行的免疫荧光共聚焦显微镜，明确证实 hFADS1 而不是 hFADS2 定位于线粒体。
