Mechanism of selective metal incorporation and cofactor maturation during tungsten cofactor biosynthesis
Final Report Abstract
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 cofactor. As molybdenum was first found to be associated with this type of pterin, it was named molybdopterin but later it became evident that tungsten-containing enzymes use the same pterin scaffold to chelate tungsten. The biosynthesis of these pterin-based metal cofactors has only been studied for molybdenum cofactors. It is commonly believed in the filed that synthesis of the pterin backbone is well conserved for both types of cofactors. However, knowledge about the incorporation of tungsten into tungsten cofactors (Wco) is lacking. Wco belongs to the family of bis-MPT cofactors whose final maturation is also poorly understood for molybdenum-containing cofactors (Moco). Therefore, the aim of this project was the functional characterization of three proteins (MoaB, MoeA1 and MoeA2) essential for metal insertion into tungsten cofactors of Pyrococcus furiosus in order to understand the molecular mechanism of Wco biosynthesis. 1. We have functionally characterized P. furiosus MoaB (PfMoaB) protein and showed catalysis of metal-binding pterin (MPT) adenylylation in the same way as E. coli MogA and plant Cnx1G. This finding demonstrated that the AMP activation of MPT prior to metal insertion is an evolutionary old and conserved step in molybdenum and tungsten cofactor biosynthesis. 2. Next we determined the crystal structure of hexameric PfMoaB and identified key residues responsible for the hexamerization. Following structure-guided mutagenesis we obtained a dimeric PfMoaB varaint, which exhibited a reduced thermal stability with a retained fold of the monomeric subunit. Interestingly, the hexamerization-deficient MoaB variant showed an increase in activity in comparison to wildtype MoaB at moderate temperatures, similar activities at 50°C and a loss of activity at hyperthermophilic temperature. These results unequivocally demonstrated that the thermal stability of PfMoaB largely depends on hexamerization. 3. In organisms dependent on molybdenum, metal insertion is catalyzed by proteins of the MoeA family. In this project we were able to demonstrate that P. furiosus MoeA1 and MoeA2 proteins, both are able to bind MPT-AMP and copurify with molybdate and tungstate. While MoeA1 showed low activity only with tungstate, MoeA2 was able to hydrolyze bound MPT- AMP with either tungstate or molybdate at equally high rates. As a result, tungsten and molybdenum cofactors were expected to released, but due to a lack of appropriate apoenzymes, we were not able to finally prove cofactor formation. 4. In the final part of the project we aimed to characterize tungsten dinucleotide cofactor formation, which however was not possible due to the lack of the MobA protein, which we could not express in a soluble and active state. However, we expressed and characterized another protein of the mob operon of P. furiosius, PfMobB, a novel member of the MobB protein family with an additional [4Fe-4S] of yet unknown function.
Publications
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2008 The function of MoaB proteins in the biosynthesis of molybdenum and tungsten cofactors. Biochemistry 47:949-56
Bevers LE, Hagedoorn, Santamaria-Araujo JA, Magalon A, Hagen W, Schwarz G
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2009 Molybdenum cofactors, enzymes and pathways Nature 460: 839-47
Schwarz G, Mendel RR, Ribbe WM
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2011 A molecular basis for tungstate selectivity in prokaryotic ABC-transport systems. J. Bacteriol. 193: 4999-5001
Bevers LE, Schwarz G & Hagen WR
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2011 Molybdenum cofactor biosynthesis in plants and humans. Coord. Chem. Rev. 255: 1145-58
Mendel RR, Schwarz G
