Pterin cofactor, substrate specificity, and observations on the kinetics of the reversible tungsten-containing aldehyde oxidoreductase fromClostridium thermoaceticum

Abstract

The αβ subunits of the tungsten-containing reversible aldehyde oxidoreductase ofClostridium thermoaceticum were shown to contain a pterin cofactor in the form of a mononucleotide. The substrate specificity of the enzyme for aliphatic and aromatic aldehydes and for carboxylates was broad. TheK m values for ethanal, propanal and butanal were 0.010–0.006 mM, but the value for methanal was 1.6 mM. Benzaldehyde derivatives with a hydroxy group in the 4-position showed millimolarK m values that were2–3 orders of magnitude higher than those of other aromatic and aliphatic aldehydes. The ratio ofk cat/Km for aldehydes and the corresponding acids is 104–105. For carboxylate reduction, 4-hydroxy benzoate again showed the highestK m value of all substrates tested. When the 4-hydroxy groups of the aldehyde and the acid were methylated, theK m values were decreased drastically. From the temperature dependence of carboxylate reduction at the expense of viologens, activation energies that depended on the substrate and on the applied viologen were calculated. The pH optima of the carboxylate reductions depended on the pK values of the acids and shifted to lower pH values with lower pK values of the acids. The ternary complex α3β3γ of the aldehyde oxidoreductase was able to dehydrogenate aldehydes to acylates with NADP+. Surprisingly the reverse reaction was observed too, although at very low rates. When exposed to air, the aldehyde oxidoreductase showed markedly enhanced lability in its reduced state compared to its oxidized state. With resting cells ofC. thermoaceticum, many carboxylates were reduced at the expense of carbon monoxide to the corresponding alcohols.