Studies on Structure and Function of Formaldehyde Dismutase

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Formaldehyde dismutase (FDM), which was found in an isolated Pseudomanas putida F61, catalyzes the dismutation of aldehydes, leading to the formation of equimolar amount of alcohols and acids. The oxidoreduction in the dismutation reaction is mediated by the coenzyme NAD(H) which firmly (but not covalently) at the active site of this enzyme. Such an enzyme that catalyzes the oxidoreduction without addition of an electron acceptor is favorable as a catalyst for a bioreactor. The kinetic parameters of the half reaction of this enzyme are very similar to those of conventional NAD-linked alcohol dehydrogenase. The most characteristic properties of FDM is the high affinity for NAD(H). In order to elucidate the protein structures participating in the binding of NAD(H), FDM gene was cloned and characterized.FDM gene was cloned onto a vector plasmid pKT230 as a 7.2 kb Sau 3Al fragment of the total DNA of P. putida F61. The gene responsible for the enzyme was recloned in pKT230 (pEC5) and ptFC19 (pEC21) as a 3.2 kb, Hind III and Pst I digest fragment. The Pseudomonas putida TN1126 harboring pEC5 (N5) exhibited the FDM activity at about 1% of the parent strain, F61. The enzyme protein was purified from the recombinant strain, N5, and obtained an electrophoretically homogeneous protein at 21% activity yield. The HPLC (TSKgel 3000SW) and SDS-PAGE proteins of the purified enzyme were identical with those of the F61 enzyme. The purified N5 enzyme exhibited only 1%. of the F61 enzyme. A part of the activity of N5 enzyme was restored by the dialysis with NAD. As to a tentative conclusion, the bacterial cells must contain a high concentration of NAD or NADH in order to synthesis of the active FDM. Determination of the base sequence of the insertion in pEC5 is in progress.