A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F 61. The enzyme catalyzes the dismutation of aldehydes and alcohol: aldehyde oxidoreduction in the absence of an exogenous electron acceptor. The enzyme is composed of four identical subunits with a M, of 44000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol. The ratio of NAD+ and NADH in a crystalline preparation of the enzyme was about 7: 3. The enzyme-bound coenzyme was completely reduced and oxidized on the addition of a large amount of an alcohol and an aldehyde respectively. Both the oxidized and reduced enzymes catalyzed the dismutation reaction to the same extent. Steady-state kinetics of the enzyme were investigated using an oxidoreduction reaction between an alcohol and p-nitroso-N,N-dimethylaniline. The enzyme obeys a ping-pong mechanism and is competitively inhibited by an alcoholic substrate analogue, pyrazole, but not coenzyme analogues, such as AMP, N-methylnicotinamide. These results indicate that NAD(H) binds firmly (but not covalently) at each active site. The enzyme-bound NAD(H) was reduced and oxidized only by the added second substrates, alcohol and aldehyde respectively, and not by exogenous electron acceptors [including NAD(H)].In the preceding studies [I -31 we found a novel enzyme, which was given the trivial name of formaldehyde dismutase, in an isolated bacterium, Pseudomonas putida F61. This enzyme catalyzed the dismutation of aldehydes (including formaldehyde), leading to the formation of equimolar amounts of the corresponding alcohols and acids. Preferable substrates,for the reaction were aldehydes that are hydrated to a great extent, such as formaldehyde, acetaldehyde and methylglyoxal. A variety of non-hydrated aldehydes was also reduced through the cross-dismutation with the hydrated aldehydes. Thus, the dismutation and cross-dismutation reactions can be seen as coupled oxidoreduction of an aldehyde (RCHO) and an 'alcohol' [RCH(OH),] formed through the hydration. Furthermore, this enzyme catalyzes the oxidoreduction of a natural alcohol and an aldehyde, leading to the exchange of the alcohol and aldehyde species. Analogous reactions with limited substrates have been reported to be catalyzed by native horse liver alcohol dehydrogenase in the presence of NAD [4-61, and by the dehydrogenase covalently bound an NAD analogue in the absence of an exogenous coenzyme [7, 81. However, formaldehyde dismutase, which is a naturally occurring bacterial enzyme, is distinct from these native and modified alcohol dehydrogenases in that it shows no requirement of an electron acceptor and there is no enhancement of its activity on the addition of an excess amount of NAD'. The oxidoreduction in the dismutation reaction is mediated by a non-dissociable coenzyme of this enzyme. In this work we report that the crystalline enzyme contains nonCorrespondence to N. Kato,
