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Tungstoenzymes are abundant among Archaea and Bacteria. The best-characterized are enzymes from Archaea. This thesis presents the characterization of a tungsten-containing aldehyde oxidoreductase (AOR) from a mesophilic bacterium for the first time. AOR from Aromatoleum aromaticum EbN1 forms a heterohexamer of three subunits in a a2b2g2 conformation. It shows a broad substrate spectrum with benzaldehyde and phenylacetaldehyde as the best tested substrates. The activity was measured with benzylviologen and NAD as well. AOR shows stability in cell extracts under oxygen exposure and pure enzyme still shows activity after hours. In contrast, AORs from Archaea lose their activity under oxygen exposure in a few minutes. Usually the AOR was purified from cells of an A. aromaticum mutant which lacks the phenylacetaldehyde dehydrogenase (Pdh) and overproduce the AOR under nitrate reducing conditions with phenylalanine as sole carbon source. As alternative, recombinantly produced AOR was purified from Aromatoleum evansii KB740 cells by affinity chromatography.
The ligation of the transition metal to the cofactor is an important step in maturation of molybdenum and tungsten enzymes. With moeA1, moeA2 and moeA3 three putative maturation factors are coded in the genome, which are related to the ligation of the metal to the pterine cofactor. All three gene products were recombinantly produced in E. coli and biochemically characterized. Additionally, the MoeA2 protein was structural analyzed. The protein MoeA2 forms a dimer and shows affinity to molybdate and tungstate as well. MoeA1 do not form a dimer and do not show affinity to W or Mo. The shorter MoeA3 forms a dimer and, based on sequence analysis, MoeA3 appears to be a putative ADP ribose pyrophosphatase. Complementation studies with different E. coli deletion mutants of genes, which are involved in the cofactor maturation, show that corresponding synthetic enzymes from A. aromaticum rescue the pterine cofactor biosynthetic pathway and recover activity of the molybdenum-dependent formate dehydrogenase H (FdhH).