Reaction mechanism and application of NAD-binding dehydrogenases

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1) Purification and properties of formaldehyde dismutase. Formaldehyde dismutase (FDM) was purified in crystalline form from Pseudomonas putida F61. The enzyme is composed of our identical subunits with a Mr of 44000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol.2) The steady-state kinetics of FDM indicates that NAD(H) binds firmly (but not covalently) at each active site, and is not replaced by exogenous NAD(H) nor the analogues. Although the substrate specificity of the enzyme seems to be similar to those of conventional NAD-linked alcohol dehydrogenase, the enzyme should be classified a novel enzyme, alcohol:aldehyde oxidoreductase (EC 1.1.99.X) on the basis of the overall reaction.3) Immobilization of FDM and conversion to methanol to formate. FDM was greatly stabilized by immobilization in a urethane prepolymer (PU-6). Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase. catalase and FDM, and with an intact cell-mixture of Hansenula polymorpha and P. putida. Furthermore, the stability of the cell-mixture was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12h. The immobilized cell-system was also effective for the conversion of several aliphatic alcohols, C_1 to C_4, to the corresponding acids.4) Utilization of NAD-binding formate dehydrogenase. NAD was covalently bound to formate dehydrogenase by a method of organic synthesis. The formate dehydrogenasegenase reactions. Among them, the enzymatic reduction of 2-oxoisocaproate to leucine was occurred stoichiometrically in the reaction system containing leucine dehydrogenase, formate-NAD complex and substrates.