Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

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Abstract
Extracts of amine-grown P. aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which were transferred to methylamine growth medium. Membrane-containing preparations from methylamine-grown cells catalyzed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. N-Methylglutamate dehydrogenase activity with phenazine methosulfate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (wt/vol) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150,000 g. Solubilization was accompanied by a change in the pH optimum for activity. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatography to yield a preparation 22-fold increased in specific activity over the crude extract. The partially-purified enzyme was active with sarcosine, N-methylalanine, N-methylaspartate and with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids and with the L-forms was obtained. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. Kinetic analysis confirmed previous observations of a group transfer (ping pong) mechanism. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. The role of the enzyme in the oxidation of methylamine is discussed.