Mechanism of selective metal incorporation and cofactor maturation during tungsten cofactor biosynthesis

在自然界中已知的三种生物活性氢化翼素中，只有一种与金属相关，从而形成一个称为钼和钨辅因子的假体基团。由于钼最初被发现与蝶呤有关，因此被命名为钼钼酸酯，但后来发现，含钨酶使用相同的蝶呤支架来螯合金属。这些基于蝶呤的金属辅助因子的生物合成只研究了钼辅助因子，但人们认为蝶呤的合成对这两种辅助因子都有很好的保守性。然而，对钨在各自辅因子中的掺入情况却一无所知。钨辅因子属于双mpt辅因子家族，其最终成熟甚至不了解含钼的同源物。因此，我们希望对金属插入到发热热球菌的钨辅因子中所必需的三种蛋白（MoaB， MoeA1和MoeA2）的功能进行表征，从而揭示钨辅因子合成的分子机制。与大肠杆菌蛋白的比较将使我们能够确定bis-MPT辅助因子合成的一般机制。此外，二甲基亚砜家族的辅因子中核苷酸的附着性尚不清楚。因此，我们将研究鸟嘌呤转移到P. furiosus甲酸脱氢酶的钨辅因子上，这将揭示双mpt鸟嘌呤二核苷酸形成的一般机制。最后，钨依赖酶将通过一个完全定义的体外系统重建。

There are three biologically active hydrogenated pterins known in nature of which only one is associated with metals thus forming a prosthetic group referred to as molybdenum and tungsten cofactors. As molybdenum was first found to be associated with the pterin it was named molybdopterin but later it became evident that tungsten-containing enzymes use the same pterin scaffold to chelate the metal. The biosynthesis of these pterin-based metal cofactors has only been studied for molybdenum cofactors but it is believed that synthesis of the pterin is well conserved for both types of cofactors. However, nothing is known about the incorporation of tungsten into the respective cofactors. Tungsten cofactors belong to the family of bis-MPT cofactors whose final maturation is not even understood for molybdenum containing homologs. Therefore, we want to characterize the functions of three proteins (MoaB, MoeA1 and MoeA2) essential for metal insertion into tungsten cofactors of Pyrococcus furiosus thereby uncovering the molecular mechanism of tungsten cofactor synthesis. Comparison to E. coli proteins will enable us to determine a general mechanism of bis-MPT cofactor synthesis. Furthermore, nucleotide attachment in cofactors of the dimethyl sulfoxide family is poorly understood. Therefore, we will investigate guanyl transfer to the tungsten cofactor of P. furiosus formate dehydrogenase, which should uncover the general mechanism of bis-MPT guanine dinucleotide formation. Finally, tungsten-dependent enzymes will be reconstituted by a fully defined in vitro system.