Hyphomicrobium strain x was grown on trimethylamine and dimethylamine as the sole sources of carbon and energy under both aerobic and anaerobic conditions and the enzymes involved in the metabolism of these compounds were investigated. During aerobic growth of the organism on trimethylamine, accumulation and subsequent utilization of dimethylamine was observed. When the organism was grown on trimethylamine under anaerobic conditions in the presence of nitrate, a sequential accumulation and utilization of dimethylamine and methylamine was found. In cell-free extracts of Hyphomicrobium x grown on trimethylamine or dimethylamine under both aerobic and anaerobic conditions the following enzyme activities were detected : trimethylamine dehydrogenase, dimethylamine dehydrogenase, y-glutamylmethylamide synthetase, N-methylglutamate dehydrogenase, methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase and hydroxypyruvate reductase. Under neither growth condition were any of the following enzyme activities detected : trimethylamine mono-oxygenase, dimethylamine monooxygenase, trimethylamine-N-oxide aldolase (demethylase) and primary-amine dehydrogenase. Trimethylamine dehydrogenase and dimethylamine dehydrogenase were partially purified from bacteria grown on dimethylamine and the results suggest that in Hyphomicrobium x a novel enzyme, namely dimethylamine dehydrogenase, participates in the oxidation of dimethylamine.
Hyphomicrobium X possesses two different enzymes for the oxidation of dimethylamine, namely dimethylamine dehydrogenase and dimethylamine mono-oxygenase. During growth of the organism in batch culture at dissolved oxygen tensions (DOT) in excess of 30 mmHg in media containing trimethylamine as the carbon and energy source, dimethylamine mono-oxygenase was the only enzyme involved in dimethylamine oxidation. The apparent K, of the mono-oxygenase for oxygen was relatively high (23-2 ,uM). The enzyme was less sensitive to inhibition by trimethylamine (& 4.2 mM) than was dimethylamine dehydrogenase (K, 7.1 ,UM) and therefore dimethylamine did not accumulate in the culture medium under these conditions. This was in contrast to observations made during anaerobic growth on trimethylamine. During growth of the organism in dimethylamine-limited chemostat cultures, the specific activities of the mono-oxygenase and the dehydrogenase were dependent on the DOT in the culture. When the DOT in the culture growing at a dilution rate of 0-10 h-l was decreased below 30 mmHg, the activity of the mono-oxygenase also decreased. In contrast, the activity of dimethylamine dehydrogenase increased, indicating that this enzyme gradually took over at the lower DOT. Below values of 5 mmHg the culture became oxygen-limited and below 3 mmHg itwas washed out. When the organism was grown at low DOT in medium supplemented with nitrate, essentially the same results were obtained, except that wash-out of the culture under anaerobic conditions did not occur. The organism was able to carry out denitrification under 'partly aerobic ' conditions
Dimethylamine dehydrogenase was purified 15.6-fold from Hyphomicrobium X grown anaerobically on dimethylamine as sole carbon source by ammonium sulphate fractionation and chromatography on DEAE-cellulose. The preparation was free from trimethylamine dehydrogenase. The molecular weight of the enzyme was 176 000 and subunit analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that it consists of two, probably identical, subunits with molecular weights of 9 1 000. 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, stoicheiometric 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 methosulphate, only phenazine ethosulphate, Wurster's blue and methylene blue served as artificial electron acceptors. The apparent K, of the enzyme for dimethylamine at pH 7-7 was 15.6 & 1.6 ,UM. Trimethylamine was a potent competitive inhibitor of dimethylamine oxidation with an apparent Ki of 7.1 ,UM. This inhibition of dimethylamine dehydrogenase by trimethylamine probably explains the observed accumulation of dimethylamine during anaerobic growth of Hyphomicrobium X on trimethylamine.
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