Dimethylamine Dehydrogenase from Hyphomicrobium X: Purification and Some Properties of a New Enzyme that Oxidizes Secondary Amines

Abstract

Dimethylamine dehydrogenase was purified 15.6-fold from Hypomicrobium X grown anaerobically on dimethylamine as sole C source by (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. The preparation was free from trimethylamine dehydrogenase. The MW of the enzyme was 176,000 and subunit analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it consists of 2, probably identical, subunits with MW of 91,000. The absorption spectrum showed a maximum at 441 nm. Reduction of the enzyme with dimethylamine produced a new absorption maximum at 356 nm, while the absorption at 441 nm decreased. The pH optimum for the oxidation of dimethylamine was 8.1. In this reaction, stoichiometric amounts of methylamine and formaldehyde were formed as products. The enzyme showed absolute specificity towards secondary amines; dimethylamine, methylethylamine, diethylamine, methylpropylamine, ethylpropylamine and methylethanolamine were oxidized while primary and tertiary amines and quaternary ammonium salts were not. Apart from phenazine methosulfate, only phenazine ethosulfate, Wurster''s blue and methylene blue served as artificial electron acceptors. The apparent Km of the enzyme for dimethylamine at pH 7.7 was 15.6 .+-. 1.6 .mu.M. Trimethylamine was a potent competitive inhibitor of dimethylamine oxidation with an apparent Ki [inhibition constant] of 7.1 .mu.M. This inhibition of dimethylamine dehydrogenase by trimethylamine probably explains the observed accumulation of dimethylamine during anaerobic growth of Hyphomicrobium X on trimethylamine.