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

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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.