Purification and properties of an amine dehydrogenase from Pseudomonas AM1 and its role in growth on methylamine.
1. Whole cells of Pseudomonas AM1 grown on methylamine oxidize methylamine, formaldehyde and formate. Crude extracts oxidize methylamine only if supplemented with phenazine methosulphate. 2. By using a spectrophotometric assay, the methylamine-oxidizing enzyme has been purified 20-fold in 31% yield. 3. The enzyme is a dehydrogenase, unable to utilize oxygen, NAD, NADP, flavines or menadione as electron acceptors, but able to utilize phenazine methosulphate, ferricyanide, cytochrome c or brilliant cresyl blue. 4. The enzyme is non-specific, readily oxidizing aliphatic monoamines and diamines, histamine and ethanol-amine. Secondary and tertiary amines, quaternary ammonium salts and aromatic amines are not oxidized. 5. The pH optima for methylamine, n-pentylamine and putrescine are respectively 7.6, 8.0 and 8.5. 6. The K(m) value for methylamine is 5.2mum and that for phenazine methosulphate 56mum. 7. The enzyme will withstand heating for 15min. at 80 degrees without loss of activity, but is inactivated at higher temperatures. It is not inactivated by any pH value between 2.6 and 10.6. 8. The dehydrogenase is inhibited by semicarbazide (K(i) 3.35mum), isoniazid (K(i) 1.17mum), cuprizone (K(i) 0.49mum), p-chloromercuribenzoate (K(i) 0.45mm) and quinacrine (K(i) 12.1mm). 9. The enzyme is absent from succinate-grown cells, and, during adaptation from succinate to methylamine, activity appears before growth on methylamine begins.
D000588:Amines / Q000378:metabolism*; D000605:Amino Alcohols / Q000378:metabolism; D002729:Chloromercuribenzoates; D002850:Chromatography, Gel; D004396:Coloring Agents; D003580:Cytochromes / Q000378:metabolism; D004579:Electron Transport; D005292:Ferricyanides / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D006632:Histamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007538:Isoniazid; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010619:Phenazines / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011796:Quinacrine; D012663:Semicarbazides; D013053:Spectrophotometry; D013386:Succinates / Q000378:metabolism; D013696:Temperature; D014812:Vitamin K / Q000378:metabolism
D000588:Amines; D000605:Amino Alcohols; D002729:Chloromercuribenzoates; D004396:Coloring Agents; D003580:Cytochromes; D005292:Ferricyanides; D005415:Flavins; D010619:Phenazines; D012663:Semicarbazides; D013386:Succinates; D009243:NAD; D014812:Vitamin K; D009249:NADP; D006632:Histamine; D010088:Oxidoreductases; D011796:Quinacrine; D010100:Oxygen; D007538:Isoniazid