Tungsten-dependent enzymes

In Chapter 4, Christian Fischer reviews catalytic properties of models and enzymes more specifically. This chapter includes a detailed kinetic evaluation of the catalytic systems. As a young academic Fischer has only recently started working in the field of molybdenum- and tungsten-dependent enzymes. He has graduated from the Leibnitz Institute of Catalysis led by Matthias Beller and is an expert in the field of catalysis. His involvement will hopefully extend what we know about the kinetics of models and enzymes continuing Holm s excellent work in this field. Notably, Fischer has already developed and investigated the first catalytic system being able to operate in pure water. 

So, what types of organisms have evolved to use tungsten-dependent rather than molybdenum-dependent enzymes, what reactions do these enzymes catalyze, and why is tungsten utilized rather than molybdenum In the following we address these questions, the answers to which are only just beginning to emerge. 

Tungsten-Dependent Aldehyde Oxidoreductase A New Family of Enzymes Containing the Pterin Cofactor... 

A family of enzymes that catalyze the molybdenum- or tungsten-cofactor dependent conversion of aldehydes to carboxylates, often using ferredoxin. See also Aldose Reductase... 

Although molybdenum and tungsten enzymes carry the name of a single substrate, they are often not as selective as this nomenclature suggests. Many of the enzymes process more than one substrate, both in vivo and in vitro. Several enzymes can function as both oxidases and reductases, for example, xanthine oxidases not only oxidize purines but can deoxygenate amine N-oxides [82]. There are also sets of enzymes that catalyze the same reaction but in opposite directions. These enzymes include aldehyde and formate oxidases/carboxylic acid reductase [31,75] and nitrate reductase/nitrite oxidase [83-87]. These complementary enzymes have considerable sequence homology, and the direction of the preferred catalytic reaction depends on the electrochemical reduction potentials of the redox partners that have evolved to couple the reactions to cellular redox systems and metabolic requirements. 

All plants depend on nitrate reductase to accomplish the seemingly trivial reaction of nitrate reduction to nitrite, often the first step of nitrogen assimilation into compounds required for growth (5, 22). Many bacteria use molybdenum or tungsten enzymes in anaerobic respiration where the terminal electron acceptor is a reducible molecule other than oxygen, such as nitrate (2, 50), polysulfide (51), trimethylamine oxide (33, 52) or dimethyl sulfoxide (DMSO) (2, 29, 30). 

The first enzyme investigated by MCD was DMSOR where the Mo center is the sole chromophore (113). The Mo(V) state of DMSOR is formed only in substoichiometric amounts, making it impossible to study its features by electronic spectroscopy or MCD. An inactive, glycerol-inhibited form of the enzyme trapped in the Mo(V) state, which was robust under the required experimental conditions, was studied instead. The resultant MCD spectrum consisted of six transitions whose behavior (temperature dependence, magnetization) mapped precisely onto the expected six transitions of a dithiolene chelated to Mo. These results were duplicated for the tungsten enzyme AOR in Pfuriosus (114). However, note that the interpretation of these results was based on the (then) prevailing hypothesis that DMSOR was a mowomolybdopterin... 

The growth of Methanobacterium wolfei is dependent on the presence of molybdenum or tungsten, and one of the two formylmethanofuran dehydrogenases from this bacterium is a tungsten enzyme (285). The reversible reaction catalyzed by these enzymes (Eq. (29)) is the first step in methane formation from CO2 in all methanogenic Archaea (286). 

Acetogenic bacteria such as Clostridium (Cl.) ther-moaceticum or Cl. formicoaceticum catalyze an NADPH2-driven CO2 reduction to formate, which is further reduced via the tetrahydrofolate pathway to the -CH3 oxidation state, as the methyl is finally incorporated into acetic acid. This anabolic formate dehydrogenase has long been known to depend on the presence of Se in the medium for its formation. The enzyme is an ai 2 heterooligomer of 340 kDa the complex contains two selenocysteine residues, two moles of a tungsten cofactor, and Fe/S centers. 

The enzyme activity is induced by both NOj" and Mo when in the presence of each other. The induction of enzyme activity by N03" is a slow process and requires mRNA-dependent synthesis of apoprotein, whereas the induction of enzyme activity by Mo is much faster, as it involves only rapid activation of the apoprotein by Mo (Jones et al., 1976, 1978). Notion and Hewitt (1979) showed that the Mo-free apoenzyme could be activated by addition of Mo complex obtained from acid washings of the native enzyme. Tungsten (W) can substitute for Mo in nitrate reductase, but the enzyme activity is decreased (Heimer, Wray, and Filner, 1969), as the formation of an active Mo cofactor is prevented. In an experiment with W-treated tobacco (Nicotiana tabacum) plants supplied with N as NOs , Deng, Moureaux, and Caboche (1989) reported a decrease in nitrate reductase activity, but several-fold increases in the accumulation of nitrate reductase apoprotein and corresponding mRNA because of excessive expression of a nitrate reductase structural gene. 

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