Formaldehyde dismutase, a novel NAD‐binding oxidoreductase from Pseudomonas putida F61

Abstract

A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F61. 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 Mr 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)]. Copyright © 1986, Wiley Blackwell. All rights reserved