Microbial Oxidation of Amines. Partial Purification of a Mixed-Function Secondary-Amine Oxidase System From Pseudomonas Aminovorans That Contains an Enzymically Active cytochrome-P-420-type Haemoprotein

Biochem J. 1971 Nov;125(2):449-59. doi: 10.1042/bj1250449.

Abstract

1. Crude extracts of Pseudomonas aminovorans grown on methylamine, di-methylamine, trimethylamine or trimethylamine N-oxide contain an enzyme or enzyme system catalysing the NADH- or NADPH- and oxygen-dependent oxidation of dimethylamine to methylamine and formaldehyde. 2. The enzyme has been partially purified about five-fold. It is unstable, but can be stabilized by addition of 5% (v/v) ethanol. 3. The partially purified enzyme will utilize either NADH (K(m) 6.5mum) or NADPH (K(m) 13.2mum): The following secondary amines have been shown to be substrates: dimethylamine, ethylmethylamine, diethylamine, methyl-n-propylamine, ethyl-n-propylamine, n-butylmethylamine and N-methylethanolamine. The K(m) values and comparative reaction rates for each substrate have been determined. Where the alkyl groups are different, the aldehyde products are derived from both groups. 4. The enzyme system has a pH optimum of 6.8 and is inhibited by mercurials, thiol compounds, cyanide and carbon monoxide. 5. The partially purified preparation had a spectral maximum at 412nm with shoulders at 427 and 550nm. Reduction with dithionite or NAD(P)H bleached the 412nm peak, and the shoulder at 427nm became a peak. Additional peaks appeared at 550 and 580-588nm. Reduction of a preparation bubbled with carbon monoxide enhanced and sharpened the Soret peak and caused it to shift to 422nm. 6. Analysis of the preparation showed the presence of flavin, acid-extractable iron and non-acid-extractable iron in the proportion 1.1:1.9:1. On reduction with dithionite or NADPH the preparation showed an electron-paramagnetic-resonance signal at around g=1.946.

MeSH terms

  • Amines
  • Carbon Monoxide
  • Cytochromes*
  • Electron Spin Resonance Spectroscopy
  • Ethylamines
  • Flavins / analysis
  • Formaldehyde
  • Hydrogen-Ion Concentration
  • Iron / analysis
  • Kinetics
  • Methylamines
  • Monoamine Oxidase / isolation & purification*
  • Monoamine Oxidase Inhibitors
  • NAD
  • NADP
  • Oxygen
  • Pseudomonas / enzymology*
  • Spectrophotometry

Substances

  • Amines
  • Cytochromes
  • Ethylamines
  • Flavins
  • Methylamines
  • Monoamine Oxidase Inhibitors
  • NAD
  • Formaldehyde
  • NADP
  • Carbon Monoxide
  • Iron
  • Monoamine Oxidase
  • Oxygen