Tungsten-dependent aldehyde oxidoreductases: specific W-cofactor insertion and interactions with Fe-S clusters in catalysis and maturation

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Aldehyde:ferredoxin oxidoreductases (AOR) belong to a family of tungsten-containing enzymes mostly known from hyperthermophilic Archaea. They contain a tungsten-bis-molybdopterin cofactor as part of the active site and are genetically and structurally distinct from the other three enzyme families of molybdopterin-containing enzymes, which mostly contain molybdenum instead of tungsten. We have recently discovered an unusually oxygen-stable AOR ortholog in the mesophilic denitrifying bacterium Aromatoleum aromaticum, which oxidises a variety of aromatic or aliphatic aldehydes to the corresponding acids. The purified protein consists of a large alpha subunit containing the W-cofactor and one Fe4S4 cluster, a small beta subunit containing four Fe4S4 clusters and a medium gamma subunit containing FAD. This complex composition and the insensitivity against oxygen differs markedly from the properties of the known archaeal orthologs and justifies a more thorough characterization of the enzyme. We intend to characterize the catalytic and structural properties of this new type of AOR isoenzyme, especially regarding the function of the apparent electron transfer relay formed by the various Fe-S clusters. The focus of these studies will be on the reversibility of the reaction and the reason for the increased oxygen stability. In addition, a recombinant expression system will be established to obtain active AOR and mutant variants, which will allow to evaluate the contributions of individual Fe-S clusters or other active site residues to the catalytic mechanism. A second main aspect of the project will be the mechanism of W-cofactor biosynthesis and incorporation into AOR. A. aromaticum synthesizes functional Mo-cofactor containing enzymes at the same time as AOR, necessitating a specific machinery of synthesis and incorporation of both cofactors into the correct enzymes. To shed more light on this process, we will study the enzymes of cofactor synthesis that are encoded in duplicated versions in the genome, particularly the Mo-/W-inserting MoeA proteins and the MoaD proteins required for introducing the SH groups of the molybdopterin cofactors. We will study the purified proteins which we will try to reconstitute into a system of in-vitro W- cofactor biosynthesis, and will also generate mutants lacking the respective genes to study their in-vivo effects. Particularly, we will investigate a hypothetical new pathway of W-cofactor maturation that involves AOR and its Fe-S clusters as scaffold system.