Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans

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Abstract
Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. The enzyme was able to catalyze the oxidation of a wide variety of primary aliphatic amines and diamines, but it did not react with secondary, tertiary, or aromatic amines. The enzyme exhibited optimal activity at pH 7.5, with Km values of 12.5 microM for methylamine and 156 microM for phenazine ethosulfate and a Vmax of 16.9 mumol/min per mg of protein. No loss of enzyme activity was observed after incubation for 48 h at pH values ranging from 3.0 to 10.5, and the enzyme was very stable to thermal denaturation. Enzyme activity and immunological detection of each subunit were only observed with cells which had been grown on methylamine as a carbon source.