Department of Biochemistry (0308), Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Biochemistry. 2010 Oct 12;49(40):8748-55. doi: 10.1021/bi100817q. Epub 2010 Sep 20.
FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.
FAD 合成酶催化将 ATP 的 AMP 部分转移到 FMN 上，生成 FAD 和焦磷酸（PP(i)）。真核生物中存在单功能 FAD 合成酶，而细菌则具有双功能酶，可催化核黄素磷酸化和 FMN 腺苷酸化生成 FAD。对古菌基因组的分析并未发现编码这两类酶的基因，但古菌中含有 FAD。我们最近发现了一种依赖于 CTP 的古菌核黄素激酶，这强烈表明存在单功能 FAD 合成酶。在此，我们报告了一种古菌 FAD 合成酶的鉴定和特性。Methanocaldococcus jannaschii 基因 MJ1179 编码一种属于核苷酸转移酶蛋白家族的蛋白质，先前被注释为甘油-3-磷酸胞苷转移酶（GCT）。克隆了 MJ1179 基因，并在大肠杆菌中异源表达了其蛋白质产物。该酶催化 FMN 与 ATP 的腺苷酸化生成 FAD 和 PP(i)。该酶来源于 MJ1179 的蛋白质已被指定为 RibL，以表明它遵循古菌 FAD 生物合成途径中的核黄素激酶（RibK）步骤。在有氧条件下分离的 RibL 仅在还原条件下才具有活性。发现 RibL 需要二价金属才能发挥活性，其中 Co(2+) 的活性最高，是 Mg(2+) 的 4 倍。该蛋白 C 末端的两个保守半胱氨酸的烷基化导致完全失活。还发现 RibL 可催化 CTP 对 FMN 的胞苷化，生成修饰的 FAD，黄素胞苷二核苷酸（FCD）。与其他 FAD 合成酶不同，RibL 不会催化从 FAD 和 PP(i) 生成 FMN 和 ATP 的逆反应。与其他 FAD 合成酶也不同，PP(i) 抑制 RibL 的活性。
